Methods and compositions for preventing and treating male erectile dysfunction and female 
sexual arousal disorder

ABSTRACT

The invention provides a method for preventing or treating male erectile dysfunction or female sexual arousal disorder by administering an effective amount of one or more factors from a group of factors including vascular endothelial growth factor, brain-derived neurotrophic factor, basic fibroblast growth factor, neurotrophin-3, neurotrophin-4, or angiopoietin-1, wherein the factor is a full length protein or a nucleic acid encoding the factor, or a functional derivative or fragment thereof, or an agent that enhances production and/or male erection or female sexual arousal stimulating function of the factor(s). Combinations, kits, and combinatorial methods are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/155,785, filed May 23, 2002, which is a continuation-in-part of U.S.patent application Ser. No. 09/909,544, filed Jul. 19, 2001, nowallowed, which claims the benefit of the priority date of U.S.provisional patent application Ser. No. 60/220,031, filed Jul. 21, 2000,under 35 U.S.C. § 119(e). The disclosures of the above-describedapplications are incorporated herein by reference in their entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention is supported in part by Grant No. DK45370 and DK51374 ofthe National Institutes of Health. The United States government may havecertain rights in this invention.

FIELD OF THE INVENTION

This invention relates generally to the field of urology. In particular,the invention provides a method for preventing or treating male erectiledysfunction or female sexual arousal disorder in a mammal in need ofsuch treatment, comprising administering an effective amount of a factorfrom a group of factors including vascular endothelial growth factor(VEGF), brain-derived neurotrophic factor (BDNF), basic fibroblastgrowth factor (bFGF), platelet-derived growth factor (PDGF),neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), or angiopoietin-1 (Ang-1),wherein the factor is a full length protein or a functional derivativeor fragment thereof, or a nucleic acid encoding said factor orfunctional derivative or fragment thereof, or an agent that enhancesproduction and/or male erection or female sexual arousal stimulatingfunction of the factor, thereby preventing or treating male erectiledysfunction or female sexual arousal disorder in the mammal.Combinations, kits, and combinatorial methods for preventing or treatingmale erectile dysfunction or female sexual arousal disorder are alsoprovided.

BACKGROUND OF THE INVENTION

VEGF is a family of proteins that were discovered on the basis of theirability to stimulate VEC (vascular endothelial cell) growth(angiogenesis). It now comprises five members, namely, VEGF-A, VEGF-B,VEGF-C, VEGF-D, and PLGF (placenta growth factor) that are encoded fromdistinct genes. Achen, et al., Proc. Nat'l. Acad. Sci. USA, 95: 548(1998), Joukov, et al., EMBO J., 15: 1571 (1996), Maglione, et al.,Oncogene, 8: 925 (1993), Olofsson, et al., Proc. Nat'l. Acad. Sci. USA,93: 2576 (1996), Yamada, et al., Genomics, 42: 483 (1997). Each of thefive members in turn comprises two or more isoforms that arise by thesplicing of their respective pre-mRNAs. For example, the VEGF-A familyincludes VEGF₂₀₆, VEGF₁₈₉, VEGF₁₈₃, VEGF₁₆₅, VEGF₁₄₅, VEGF₁₂₁, andVEGF₁₁₁. Anthony, et al., Placenta, 15: 557 (1994), Neufeld, et al.,FASEB J., 13: 9 (1999), Lei, et al., Biochim. Biophys. Acta, 1443: 400(1998), Jingjing, et al., Ophthamol. Vis. Sci., 40: 752 (1999), Cheung,et al., Am. J. Obstet. Gynecol., 173: 753 (1995), Burchardt, et al.,Biol. Reprod., 60: 398 (1999). Among all VEGF proteins and isoforms,VEGF₁₆₅ is by far the most frequently used form of VEGF both in basicand clinical studies.

It has been shown that, among different vascular cell types(endothelial, smooth muscle cells (SMC), and fibroblasts), SMC is theprincipal source for the secreted VEGF. Pueyo, et al., Exp. Cell Res.,238: 354 (1998). Expression of VEGF in SMC is upregulated by multiplefactors including phorbol esters (Tischer, et al., J. Biol. Chem., 266:11947 (1991)), cAMP (Claffey, et al., J. Biol. Chem., 267: 16317(1992)), and hypoxia (Goldberg, et al., J. Biol. Chem., 269: 4355(1994), Shweiki, et al., Proc. Nat'l Acad. Sci. USA, 92: 768 (1995)).The secreted VEGF acts on VEC principally through two different cellsurface receptors, VEGFR-1 and VEGFR-2. Activation of VEGFR-1 results inVEC migration, while activation of VEGFR-2 VEC migration andproliferation. Waltenberger, et al., J. Biol. Chem., 269: 26988 (1994),Neufeld, et al., FASEB J., 13: 9 (1999), Ortega, et al., Front. Biosci.,4: D141 (1999). Although VEGFR-1 and VEGFR-2 have long been consideredendothelium-specific, they have both been detected in human uterine andbovine aorta SMC. Grosskreutz, et al., Microvasc. Res., 58: 128 (1999),Brown, et al., Lab. Invest., 76: 254 (1997). Cultured uterine SMCresponded to VEGF in the form of cell proliferation and cultured aortaSMC cell migration. Cultured human colon SMC, however, did not expressVEGF receptors, nor did they respond to VEGF treatment. Brown, et al.,Lab. Invest., 76: 254 (1997).

Angiogenesis is a complex process that includes activation, migrationand proliferation of endothelial cells and formation of new bloodvessels. D'Amore, et al., Ann. Rev. Physiol., 49(9-10): 453-64 (1987).VEGF has been shown to be intimately involved in the entire sequence ofevents leading to growth of new blood vessels. Gross, et al., Proc.Nat'l. Acad. Sci., 80(9): 2623-27 (1983), Folkman, et al., Proc. Nat'l.Acad. Sci., 76(10): 5217-21 (1979). Five human VEGF isoforms of 121,145, 165, 189 and 206 amino acids have been isolated. Gross, et al.,Proc. Nat'l. Acad. Sci., 80(9): 2623-27 (1983), Leung, et al., Science,246: 1306-09 (1989), Poltorak, et al., J. Biol. Chem., 272(11): 7151-78(1997). Among the isoforms, VEGF 165 seems to be the most effective andmost commonly used. The effect of VEGF 165 in augmenting perfusion andin stimulating formation of collateral vessels has been shown in animalmodels Hopkins, et al., J. Vascular Surgery, 27(5): 886-94 (1998),Asahara, et al., Circulation, 91(11): 2793-801 (1995), Hariawala, etal., J. Surg. Res., 63(1): 77-82 (1996), Bauters, et al., Circulation,91(11): 2802-9 (1995), Bauters, C., et al., Am. J. Physiol., 267(4 Pt2): H1263-71 (1994), Takeshita, et al.,. J. Clin. Invest., 93(2): 662-70(1994), Takeshita, et al., Circulation, 90(5 Pt 2): II228-34 (1994),Takeshita, et al., Am. J. Path., 147(6): 1649-60 (1995), Banai, et al.,Circulation, 89(5): 2183-9 (1994). In clinical trials, successfulinduction of collateral blood vessels in ischemic heart disease andcritical limb ischemia by VEGF have also been reported. Baumgartner, etal., Circulation, 97(12): 1114-23 (1998), Losordo, et al., Am. Heart J.,138(2Pt2): 132-41 (1999).

Platelet-derived growth factor (PDGF) is a potent mitogen for cells ofmesenchymal origin, stimulating both connective tissues and neuroglialcells. PDGF also acts as a potent chemoattractant for mesenchymal cells,mononuclear cells, and neutrophils. PDGF is stored in platelet granulesand released with platelet activation. Other cell types also producePDGF, including endothelial cells, monocytes/macrophages, vascularsmooth muscle cells, fibroblasts, and cytotrophoblasts. PDGF consists ofdisulfide-linked dimers of αα, αβ, or ββ configuration. PDGF has a shorthalf-life and usually produces only local effects. Two distinct PDGFreceptors have been identified that are structurally related and have anintracellular protein kinase domain.

Neurotrophins are a class of structurally related growth factors thatpromote neural survival and differentiation. They stimulate neuriteoutgrowth, suggesting that they can promote regeneration of injuredneurons, and act as target-derived neurotrophic factors to stimulatecollateral sprouting in target tissues that produce the neurotrophin.Korsching, J. Neurosci., 13: 2739 (1993). Recently, local synthesis andautocrine mechanisms of action have been reported. Lewin and Barde, Ann.Rev. Neurosci., 19: 289 (1996). In vivo overexpression of a neurotrophicfactor, through gene transfer, would ensure local and continuousneurotrophin production in a manner resembling the physiologic, as theseproteins are usually produced and secreted by target and glial cellssurrounding neurons.

Neurotrophin-3 (NT-3) is a member of the neurotrophin class ofstructurally related growth factors. NT-3 is a 27 kDa homodimer thatsupports the growth and survival of sympathetic neurons as well assensory neurons. NT-3 is highly conserved across species and isprimarily expressed in kidney, spleen, and heart with lower expressionlevels found in the skin, skeletal muscle, lung, thymus, and ovaries.NT-3 binds the low affinity NGF receptor, p75^(NTR), and may initiateapoptosis through this receptor. NT-3 also binds and induces signalingthrough the TrkC receptor.

Neurotrophin-4 (NT-4) is yet another member of this class ofneurotrophins. NT-4 is a homodimer that supports the growth and survivalof sympathetic neurons, dorsal root ganglion neurons, nodose ganglionneurons, basal forebrain cholinergic neurons and neurons of the locuscoeruleus. NT-4 is less highly conserved between species, unlike otherneurotrophins. NT-4 expression is widespread in brain and peripheraltissues. NT-4 induces cellular signaling through the p75^(NTR) receptoras well as the TrkB receptor.

Brain-derived neurotrophic factor (BDNF), another member of theneurotrophins, was initially characterized as a basic protein present inbrain extracts and capable of increasing the survival of dorsal rootganglia. Leibrock, et al., Nature, 341: 149 (1989). When axonalcommunication with the cell body is interrupted by injury, Schwann cellsproduce neurotrophic factors such as nerve growth factor (NGF) and BDNF.Neurotrophins are released from the Schwann cells and disperseddiffusely in gradient fashion around regenerating axons, which thenextend distally along the neurotrophins' density gradient. Ide,Neurosci. Res., 25: 101 (1996). Local application of BDNF to transectednerves in neonatal rats has been shown to prevent the massive death ofmotor neurons that follows axotomy. DiStefano, et al., Neuron, 8:983(1992), Oppenheim, et al., Nature, 360: 755 (1992), Yan, et al., Nature,360: 753 (1992). The mRNA titer of BDNF increases to several times thenormal level 4 days after axotomy and reaches its maximum at 4 weeks.Meyer, et al., J. Cell Biol., 119: 45 (1992). Moreover, BDNF has beenreported to enhance the survival of cholinergic neurons in culture.Nonomura, et al., Brain Res., 683: 129 (1995).

Angiopoietin-1 (Ang-1) is a member of a family of endothelium growthfactors. Ang-1 is a ligand for the Tie-2 receptor, a receptor tyrosinekinase with immunoglobulin and epidermal growth factor homology domainsexpressed primarily on endothelial cells and very early hematopoieticcells. Ang-1 promotes chemotaxis, cell survival, cell sprouting, vesselgrowth and stabilization of Tie-2-expressing endothelial cells. Ang-1 isthought to have a distinct angiogenic role from that of VEGF involvingthe recruitment of peri-endothelial cells that will become pericytes andsmooth muscle tissue of the blood vessel, thereby maintaining thestability of the blood vessels. See, e.g., Hanahan, D., Science, 277:48-50.

Basic fibroblast growth factor (bFGF) is a member of the fibroblastgrowth factor family. bFGF stimulates the proliferation of all cells ofmesodermal origin including smooth muscle cells, neuroblasts, andendothelial cells. bFGF stimulates neuron differentiation, survival, andregeneration. In vitro functions suggest that bFGF modulatesangiogenesis, wound healing and tissue repair, and neuronal function invivo. bFGF, a heparin-binding growth factor, is capable of inducingfunctionally significant angiogenesis in models of myocardial and limbischemia. Zheng, et al., Am. J. Physiol. Heart Circ. Physiol., 280:H909-17 (2001), Laham, et al., J. Am. Coll. Cardiol., 36: 2132-39(2000), Laham, et al., Curr. Interv. Cardiol. Rep., 1: 228 (1999),Unger, et al., Am. J. Cardiol., 85: 1414-19 (2000), Kawasuji, et al.,Ann. Thorac. Surg., 69: 1155 (2000), Rajanayagam, et al., J. Am. Coll.Cardiol., 35: 519 (2000), Kornowski, et al., Circulation, 101: 545-48(2000), Ohara, et al., Gene Ther., 8: 837 (2001), Lazarous, et al., J.Am. Coll. Cardiol., 36: 1239 (2000), Rakue, et al., Japan Circ. J., 62:933-39 (1998), Baffour, et al., J. Vasc. Surg., 16: 181 (1992).

Erectile function is a hemodynamic process of blood in-flow and pressuremaintenance in the cavernosal spaces. Christ, Urol. Clin. North Am., 22:727 (1995). Following sexual arousal and the release of nitric oxide tothe erectile tissue, three processes occur to achieve an erection. Theseare relaxation of the trabecular smooth muscle, arterial dilation andvenous compression. Id. During this final stage, arterial flow fillssinusoidal spaces, compressing subtunical venules thereby reducingvenous outflow. Blood flows into the cavernous spaces of the penis, thusexpanding and stretching the penis into a rigid organ. The flow of bloodin and out of the cavernous spaces is controlled by cavernous smoothmuscle cells (CSMC) embedded in the trabeculae of the cavernous spaces.With normal erectile function, a high intracavernous pressure (ICP) ismaintained with a low inflow rate. Karadeniz, et al., Urol. Int., 57: 85(1996).

As such, the penis is a predominantly vascular organ, and vascular orpenile arterial insufficiency is the most common etiology of erectiledysfunction (ED). Sinusoidal smooth muscle atrophy and collagendeposition is a common finding in men with long standing ED of variousetiologies, whether due to hormonal, neurological or vascular causes.Karadeniz, et al., Urol. Int., 57: 58 (1996). Such degradation in smoothmuscle quantity and quality leads to veno-occlusive dysfunction. Thisrepresents an end-stage muscular degeneration akin to myocardial changeswith congestive heart failure or dilated cardiomyopathy for which notreatment currently exists with hope of reversing the underlyingpathologic process.

Veno-occlusive disease is a common finding among patients with erectiledysfunction (ED). Following radical prostatectomy, for example,approximately 30% of patients may have vasculogenic ED in addition toneurogenic ED and at least half of these men may have venous leak.Regardless of the etiology of organic ED (neurogenic, traumatic,hormonal, and vascular, etc.), venous leakage is a common finalcondition resulting from smooth muscle atrophy. Mersdorf, et al., J.Urol., 154: 749 (1991). Veno-occlusive dysfunction is the most commonetiology of ED among non-responders to medical management of ED. None ofthe medical therapy currently exists is curative for this condition.Patients with veno-occlusive dysfunction exhibit a poor response tointracavernous injection with vasoactive agents (papavarine,prostaglandin E1, phentolamine, or combinations, for example), despitegood arterial flow demonstrated by duplex ultrasound. The diagnosis ofveno-occlusive disease may be confirmed with specific findings oncavemosometry and cavemosography. Nehra, et al., J. Urol., 156:1320(1996).

Atherosclerotic or traumatic arterial occlusive disease of thepudendal-cavernous-helicine arterial tree can decrease the perfusionpressure and arterial flow to the sinusoidal spaces, thus decreasing therigidity of the erect penis. Common risk factors associated withgeneralized arterial insufficiency include hypertension, hyperlipidemia,cigarette smoking, diabetes mellitus, and pelvic irradiation. Goldstein,et al., JAMA, 251: 903-910 (1984), Rosen, M. P., et al., Radiology,174(3 Pt 2): 1043-48 (1990), Levine, F. J., et al., J. Urology, 144(5):1147-53 (1990). Epidemiological studies have shown a high incidence ofED in patients with coronary arterial disease. Heaton, J. P., et al.,Int'l J. Impotence Res., 8(1): 35-39 (1996). Focal lesion of the commonpenile or cavernous artery is most often seen in young patients who havesustained blunt pelvic or perineal trauma such as in cases of bikingaccidents. Levine, F. J., et al., J. Urology, 144(5): 1147-53 (1990).

Because of the close proximity of the cavernous nerves to the capsule ofthe prostate, ED is a frequent complication after radical prostatectomyor cystectomy and prostatic cryosurgery. Although the nerve-sparingprostatectomy technique developed by Walsh, et al., Br. J. Urol., 56:694 (1984) has significantly reduced the postoperative impotence rate, alarge number of patients still suffer from inadequate penile rigidity.Peripheral nerve regeneration is a slow process, and the fact that mostpatients do not recover potency for 6 months to 2 years indicatessubstantial axonal damage, even with preservation of the neural sheath.An anatomic study of the cavernous nerves by Paick et al., Urology, 42:145 (1993) revealed both a medial and a lateral bundle of cavernousnerves at the level of the prostate, suggesting that in some cases thelateral bundle can be saved, even in non-nerve-sparing prostatectomy.

The sprouting of the remaining nerves in penile tissue appears to bemore important in regeneration than re-growth of nerves through thedamaged and fibrotic tissues. The importance of sprouting in theremaining nerves was confirmed in an animal study that revealedregeneration of the cavernous nerves after unilateral resection.Carrier, et al., J. Urol., 153: 1722 (1995). In addition, a previousstudy in our laboratory showed that systemic growth hormone injectionsignificantly enhanced cavernous nerve regeneration after unilateralinjury. Jung, et al., J. Urol., 160: 1899(1998).

Methods for treating erectile dysfunction have included from theadministration of prostaglandin E (U.S. Pat. No. 5,942,545), localadministration of vascular muscle relaxants and vasoactivepharmaceutical agents. See, for example, U.S. Pat. Nos. 5,942,545,6,056,966; and 5,646,181.

Advancement in molecular biology has brought improved understanding ofpathophysiology on the gene and molecular level, and offers promise oftreatment possibilities aimed at a specific pathologic molecularmechanism. As in other vasculopathies such as limb claudication(Baumgartner, et al., Circulation, 97: 1114 (1998) and coronary arterydisease (Symes, et al., Ann. Thorac. Surg., 68: 830 (1999), treatmentwith VEGF in either protein or gene form has increased neovascularity inanimal models and improved symptomatic angina and wound healing inhumans with inoperable heart disease and critical limb ischemia,respectively. The penis represents a convenient tissue target for geneor growth factor therapy due to the penis' external location on thebody, ubiquity of endothelial-lined spaces and low-level blood flow inthe flaccid state. In addition, the penis is filled with billions ofendothelial and smooth muscle cells both are rich in VEGF receptors.Liu, et al., J. Urol., 166: 354-360 (2001).

Recently, we have established an animal model in which CSMC was seendecreased following internal iliac artery ligation that restricted bloodsupply to the penis. However, rats treated with intracavernous injectionof vascular endothelial growth factor (VEGF) shortly after internaliliac artery ligation had nearly normal CSMC. The protective effects ofVEGF on CSMC could be due to partial restoration of blood supply as VEGFis expected to stimulate vascular endothelial cell (VEC) proliferation.Lin, et al., Proc. Nat'l Acad. Sci. USA, 97: 10242-47 (2000).Alternatively, VEGF might act directly on CSMC, as we will presentevidence that CSMC express one of the two principal VEGF receptors.Sondell, et al., Eur. J. Neurosci., 12: 4243-54 (2000); Liu, et al., J.Urol., 166: 354-360 (2001).

Females can also have sexual dysfunction, and this dysfunction canincrease with age. It is usually associated with the presence ofvascular risk factors, genital smooth muscle atrophy, and onset ofmenopause. Some of the vascular and muscular mechanisms that contributeto penile erection in the male are believed to be similar vasculogenicfactors in the female genital response. It is known that in women sexualarousal is accompanied by arterial inflow which engorges the vagina andincreases vaginal lubrication, and that the muscles in the clitoris andthe perineum assist in achieving clitoral erection.

In the female patient, sexual arousal disorder can arise from organicand pyschogenic causes, or from a combination of the foregoing. Femalesexual arousal disorder is classified into five categories: 1)hypoactive sexual desire disorder, 2) sexual aversion disorder, 3)sexual arousal disorder, 4) orgasmic disorder, and 5) sexual paindisorder. The present invention applies to sexual arousal disorder.Sexual arousal disorder is the persistent or recurring inability toattain or maintain adequate sexual excitement, causing personaldistress. It may be experienced as the lack of subjective excitement orthe lack of genital lubrication or swelling or other somatic responses.Organic female sexual arousal disorder is known to be related in part tovasculogenic impairment resulting in inadequate blood flow, vaginalengorgement insufficiency and clitorial erection insufficiency. Animalstudies have demonstrated the dependence of vaginal vascular engorgementand clitoral erection on blood flow. See, for example, Park et al.,“Vasculogenic female sexual dysfunction: the hemodynamic basis forvaginal engorgement insufficiency and clitoral erectile insufficiency,“Int'l J. Impotence Res., 9(1), 27-37 (March 1997).

Female sexual dysfunction has been treated with pharmacologicalintervention to stimulate blood flow as well as with prostaglandins.See, for example, U.S. Pat. Nos. 6,193,992 B1; 5,945,117; 6,031,002; and5,891,915.

SUMMARY OF THE INVENTION

The present invention provides for methods, combinations, and kits forthe treatment and prevention of male erectile dysfunction or femalesexual arousal disorder. These methods, combinations, and kits involvethe administration of vascular endothelial growth factor (VEGF),brain-derived growth factor (BDNF), basic fibroblast growth factor(bFGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), platelet-derivedgrowth factor (PDGF), angiopoietin-1 (Ang-1), or a combination thereof.

In one aspect, the present invention provides for a method forpreventing or treating male erectile dysfunction or female sexualarousal disorder, which method comprises administering to a mammal towhom such prevention or treatment is needed or desirable, an effectiveamount of a factor, wherein the factor is VEGF, BDNF, bFGF, NT-3, NT-4,PDGF, Ang-1, an agent that enhances production and/or male erection orfemale sexual arousal stimulating function of the factor, or acombination thereof, thereby preventing or treating the male erectiledysfunction or the female sexual arousal disorder in the mammal.Preferably, the factor is a full length protein or a functionalderivative or fragment thereof or a nucleic acid encoding the factor orfunctional derivative or fragment thereof.

In a specific embodiment, the mammal is a human and the factor, or afunctional derivative or fragment thereof, or the nucleic acid encodingthe factor, or a functional derivative or fragment thereof, is of humanorigin.

Preferably, the factor protein or nucleic acid, or a functionalderivative or fragment thereof, is administered by intracavernousinjection, subcutaneous injection, intravenous injection, intramuscularinjection, intradermal injection, or topical administration.

In a specific embodiment, the factor nucleic acid, or a functionalderivative or fragment thereof, is administered via a gene therapyvector, preferably the gene therapy vector is an adenovirus associatedvector, a retroviral vector, an adenovirus vector, or a lentivirusvector. More preferably, the gene therapy vector is an adenovirusassociated vector.

In yet another specific embodiment, the factor protein, or a functionalderivative or fragment thereof, is administered via a liposome.

In another specific embodiment, the factor nucleic acid, or a functionalderivative or fragment thereof, is administered via a liposome.

Preferably, the male erectile dysfunction to be treated or prevented iserectile dysfunction induced by or secondary to nerve dysfunction,arterial insufficiency, venous leakage, severe vascular insufficiency,mild vascular disease, hormonal insufficiency, drug use, surgery,chemotherapy, or radiation.

Preferably, the female sexual arousal disorder to be treated orprevented is sexual dysfunction induced by or secondary to nervedysfunction, arterial insufficiency, severe vascular insufficiency, mildvascular disease, hormonal insufficiency, drug use, surgery,chemotherapy, or radiation.

In a specific embodiment, the factor protein or a functional derivativeor fragment thereof, or a nucleic acid encoding the factor or functionalderivative or fragment thereof, or an agent that enhances productionand/or female sexual arousal stimulating function of the factor, isadministered in an amount sufficient to improve blood flow andregenerate nerve and smooth muscle in the clitoris and vaginal wall.

In another specific embodiment, the factor protein or a functionalderivative or fragment thereof, or a nucleic acid encoding the factor orfunctional derivative or fragment thereof, or an agent that enhancesproduction and/or female sexual arousal stimulating function of thefactor, is administered in a cream or via injection to the clitoris andvaginal wall of the patient.

In yet another specific embodiment, the factor protein or a functionalderivative or fragment thereof, or a nucleic acid encoding the factor orfunctional derivative or fragment thereof, or an agent that enhancesproduction and/or the male erection or female sexual arousal stimulatingfunction of the factor, is administered by intracavernous injection.

In a preferred embodiment, the male erectile dysfunction or femalesexual arousal dysfunction is induced by or secondary to nerve injury,and the combination of factors administered are: a) VEGF and NT-3, b)VEGF and NT-4, or c) VEGF and BDNF.

In another preferred embodiment, the male erectile dysfunction or femalesexual arousal disorder is induced by or secondary to severe vascularinsufficiency and the combination of factors administered are: a) VEGFand PDGF, b) VEGF and bFGF, or c) VEGF and Ang-1.

In yet another preferred embodiment, the male erectile dysfunction orfemale sexual arousal dysfunction is induced by or secondary to mildvascular disease and the factor administered is VEGF.

In another aspect, the present invention provides for a combination forpreventing or treating male erectile dysfunction or female sexualarousal disorder, which combination comprises: a) an effective amount ofan agent that stimulates male erectile or female sexual function; and b)an effective amount of a factor, wherein the factor is a full lengthprotein or a functional derivative or fragment thereof, or a nucleicacid encoding said factor or functional derivative or fragment thereof,an agent that enhances production and/or male erection or female sexualarousal stimulating function of said factor, or a combination thereof,and the factors include VEGF, BDNF, bFGF, NT-3, NT-4, PDGF, and Ang-1.

In yet another aspect, the present invention provides a combination forpreventing or treating male erectile dysfunction or female sexualarousal disorder induced by or secondary to nerve injury, whichcombination comprises: a) an effective amount of VEGF, wherein the VEGFis a full length protein or a functional derivative or fragment thereof,or a nucleic acid encoding the VEGF or functional derivative or fragmentthereof; and b) an effective amount of a factor selected from the groupconsisting of NT-3, NT-4, and BDNF, wherein the factor is a full lengthprotein or a functional derivative or fragment thereof, or a nucleicacid encoding the factor or functional derivative or fragment thereof,or an agent that enhances production and/or male erection or femalesexual arousal stimulating function of the factor.

In another aspect, the present invention provides a combination forpreventing or treating male erectile dysfunction or female sexualarousal disorder induced by or secondary to severe vascularinsufficiency, which combination comprises: a) an effective amount ofVEGF, wherein the VEGF is a full length protein or a functionalderivative or fragment thereof, or a nucleic acid encoding the VEGF orfunctional derivative or fragment thereof, and b) an effective amount ofa factor selected from the group consisting of PDGF, bFGF, and Ang-1,wherein the factor is a full length protein or a functional derivativeor fragment thereof, or a nucleic acid encoding the factor or functionalderivative or fragment thereof, or an agent that enhances productionand/or male erection or female sexual arousal stimulating function ofthe factor.

In yet another aspect, the present invention provides for theabove-described combinations, preferably in the form of a pharmaceuticalcomposition, that can be used for preventing or treating male erectiledysfunction or female sexual arousal disorder. The above-describedcombinations can further comprise a pharmaceutically acceptable carrieror excipient.

In another aspect, the present invention provides a method forpreventing or treating male erectile dysfunction or female sexualarousal disorder, which method comprises administering an effectiveamount of the above described combinations to a mammal in need thereof,thereby preventing or treating the male erectile dysfunction or thefemale sexual arousal disorder in the mammal.

In one aspect, the present invention provides for a kit comprising theabove-described combinations and an instruction for using thecombination in treating or preventing male erectile dysfunction orfemale sexual arousal disorder.

In yet another aspect, the present invention provides a method ofpromoting sprouting of new nerve fibers from blood vessel explants,which method comprises the steps of: a) isolating a blood vessel; b)attaching the blood vessel to a media-coated coverslip; and c)incubating with a growth-stimulating compound. In one preferredembodiment, the growth-stimulating compound is VEGF. The presentinvention also provides for a method of identifying a compound forpromoting sprouting of new nerve fibers from blood vessel explants,which method comprises assaying candidate compounds for nerve growthpromoting activity ex vivo using the above described method andidentifying a compound that promotes nerve growth in a blood vesselexplant as indicative of a compound that promotes nerve growth.

In another aspect, the present invention provides for a method ofinducing angiogenesis, which method comprises the steps of: a)co-culturing an isolated blood vessel and an isolated muscle explant;and b) incubating with a growth-stimulating compound. In a preferredembodiment, the compound is a combination of VEGF and PDGF. The presentinvention also provides for a method of identifying a compound forinducing angiogenesis, which method comprises assaying candidatecompounds for angiogenic activity ex vivo using the method describedabove, and identifying a compound that promotes angiogenesis in a bloodvessel cell as indicative of a compound that promotes angiogenesis.

In yet another aspect, the present invention provides for a method forpromoting growth of cavernous nerves from major pelvic ganglia (MPG),which method comprises contacting the MPG with an effective amount of afactor, wherein the factor is selected from the group consisting ofvascular endothelial growth factor (VEGF), brain-derived growth factor(BDGF), basic fibroblast growth factor (bFGF), neurotrophin-3 (NT-3),neurotrophin-4 (NT-4), platelet-derived growth factor (PDGF), andangiopoietin-1 (Ang-1), thereby promoting growth of the cavernous nervesfrom the MPG.

In another aspect, the present invention provides for a method ofidentifying a compound that promotes growth of cavernous nerves frommajor pelvic ganglia (MPG), which method comprises: a) in vitroculturing MPG; b) measuring growth of cavernous nerves from the MPG inthe presence and absence of a candidate compound; and c) identifying acompound that promotes nerve growth as indicative of a compound thatpromotes growth of cavernous nerves from MPG.

Other aspects of the invention are described throughout thespecification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Electrostimulation of the cavernous nerve at 8 weeks: A) shamoperation group; B) LacZ group; and C) BDNF group. Note higher maximalintracavernous pressure in the BDNF than in the LacZ group. Scanrate=10/sec.

FIG. 2. Quantification of VEGF secreted by CSMC. Equal number (4×10⁵) ofCSMC from different-aged rats were seeded in each well of 6-well platesand allowed to grow for three days in medium containing 10% of FBS. Themedium from each well was then assayed for the concentration ofrat-specific VEGF (panel B) and the cell number was determined (panelA). The calculated amount of VEGF in each well was then divided by thenumber of cells in each well to derive the data shown in panel C.

FIG. 3. Effects of VEGF on the growth rates of VSMC and CSMC. Equalnumbers (5,000) of VSMC (from 16-weeks-old rats, panel A) and CSMC (fromrats of indicated ages, panels B to I) were seeded in each well of96-well plates and allowed to grow for three days in media containingthe indicated concentrations of VEGF. The final numbers of cells weredetermined with a proliferation assay kit and expressed as opticaldensity (OD_(490nm)) values. These values were converted into % ofcontrol (shown on the left of panels A through J) with the value of thegrowth rate in medium containing no added VEGF being referred to ascontrol (i.e., 100%). Panel J compares the growth rates of CSMC fromrats of the indicated ages in media containing the optimal concentration(12.5 ng/ml) of VEGF.

FIG. 4. Effects of VEGF on the mobility of VSMC and CSMC. Equal numbers(8,000) of VSMC (from 16-weeks-old rats, panel A) and CSMC (from rats ofindicated ages, panels B to I) were loaded in each well of 24-wellTranswell plates and allowed to migrate through a membrane toward amedium containing the indicated concentration of VEGF. Four hours later,the numbers of cells that had migrated through the membrane were countedunder a microscope. These numbers are indicated on the left of thepanels. Panel J compares the numbers of CSMC (from rats of the indicatedages) that had migrated through the membrane toward media containing theoptimal concentration (10 ng/ml) of VEGF.

FIG. 5. Identification of VEGFR-1 and VEGFR-2 mRNA expression. RNAs ofthe following cells and tissues (lanes 1-15) were subjected to RT-PCRwith primer pair VEGFR-1s and VEGFR-1a, primer pair VEGFR-2s andVEGFR-2a, and primer pair β-actin-s and β-actin-a (Table 1). Thereaction products were electrophoresed in a 1.5% agarose gel and stainedwith ethidium bromide. The RT-PCR product in each lane was derived from25 ng (for VEGFR-1 and VEGFR-2) or 1 ng (for β-actin) of total cellularor tissue RNAs. M, 100-bp size marker. Lane 1, CSMC from 1-week-oldrats; lane 2, CSMC from 2-weeks-old rats; lane 3, CSMC from 3-weeks-oldrats; lane 4, CSMC from 4-weeks-old rats; lane 5, CSMC from 6-weeks-oldrats; lane 6, CSMC from 11-weeks-old rats; lane 7, CSMC from16-weeks-old rats; lane 8, CSMC from 28-months-old rats; lane 9, aortaSMC from 16-weeks-old rats; lane 10, heart of a 16-weeks-old rat; lane11, aorta of a 16-weeks-old rat; lane 12, corpus cavernosum of a16-weeks-old rat.

FIG. 6. Identification of VEGFR-1 protein expression. Protein extractsof CSMC from rats of the following ages (lanes 1-8) were electrophoresedin 7.5% SDS-PAGE and then transferred to PVDF membrane. Detection ofVEGFR-1 protein on the membrane was performed by the ECL procedure usingan anti-VEGFR-1 rabbit serum. Lane 1, 1-week-old; lane 2, 2-weeks-old;lane 3, 3-weeks-old; lane 4, 4-weeks-old; lane 5, 6-weeks-old; lane 6,11-weeks-old; lane 7, 16-weeks-old; lane 8, 28-months-old.

DETAILED DESCRIPTION OF INVENTION

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, patentapplications and other publications and sequences from GenBank and otherdatabases referred to herein are incorporated by reference in theirentirety. If a definition set forth in this section is contrary to orotherwise inconsistent with a definition set forth in patents, patentapplications and other publications and sequences from GenBank and otherdatabases that are herein incorporated by reference, the definition setforth in this section prevails over the definition that is incorporatedherein by reference.

As used herein, “a” or “an” means “at least one” or “one or more.”

As used herein, an “erectile dysfunction (or impotence)” refers to theinability of a male mammal, e.g., a man, to achieve and maintain penileerection for satisfactory sexual intercourse.

As used herein, a “female sexual arousal disorder” refers to thepersistent or recurring inability to attain or maintain adequate sexualexcitement causing personal distress. It may be experienced as lack ofsubjective excitement or lack of genital (lubrication and swelling) orother somatic responses.

As used herein, “vascular endothelial growth factor (VEGF)” or“brain-derived neurotrophic factor (BDNF)” or “basic fibroblast growthfactor (bFGF)” or “platelet-derived growth factor (PDGF)” or“neurotrophin-3 (NT-3)” or “neurotrophin-4 (NT-4)” or “angiopoietin-1(Ang-1)” includes those variants with conservative amino acidsubstitutions that do not substantially alter their male erection- orfemale sexual arousal-stimulating activity. Suitable conservativesubstitutions of amino acids are known to those of skill in this art andmay be made generally without altering the biological activity of theresulting molecule. Those of skill in this art recognize that, ingeneral, single amino acid substitutions in non-essential regions of apolypeptide do not substantially alter biological activity (see, e.g.,Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, TheBejacmin/Cummings Pub. co., p. 224).

As used herein, a “functional derivative or fragment of a factor” refersto a derivative or fragment of the factor that still substantiallyretains its function as an erection or sexual arousal stimulant.Normally, the derivative or fragment retains at least 50% of itserection or sexual function stimulating activity. Preferably, thederivative or fragment retains at least 60%, 70%, 80%, 90%, 95%, 99% and100% of its erection or sexual function stimulating activity.

As used herein, an “agent that enhances production of the factor” refersto a substance that increases transcription and/or translation of afactor gene, or a substance that increases post-translationalmodification and/or cellular trafficking of a factor precursor, or asubstance that prolongs half-life of a factor protein.

As used herein, an “agent that stimulates erection or female sexualarousal function activity of a factor” refers to a substance thatincreases potency of the factor's erection or sexual arousal stimulatingactivity, or a substance that increases sensitivity of a factor'snatural ligand in an erection or sexual arousal stimulation signalingpathway, or a substance that decreases potency of a factor's antagonist.Such an agent is not VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1.

As used herein, a “combination” refers to any association between two oramong more items.

As used herein, a “composition” refers to any mixture of two or moreproducts or compounds. It may be a solution, a suspension, liquid,powder, a paste, aqueous, non-aqueous or any combination thereof.

As used herein, a “nerve dysfunction” refers to the inability of thepenis to hold the blood during erection or the persistent or recurringinability to attain or maintain adequate sexual excitement in a male orfemale mammal causing personal distress including, but not limited todysfunction caused by diabetes mellitus, hypertension, hyperlipidemia,penile injury, aging, pelvic surgery or irradiation.

As used herein, an “arterial insufficiency” refers to reduced perfusionpressure and arterial flow associated with trauma or disease including,but not limited to, that associated with hypertension, hyperlipidemia,cigarette smoking, diabetes mellitus, and pelvic irradiation.

As used herein, a “venous leakage” refers to the inability of the penisto hold the blood during erection caused by a disorder including, butnot limited to diabetes mellitus, hypertension, hyperlipidemia, penileinjury, aging, pelvic surgery or irradiation.

As used herein, a “hormonal insufficiency” refers to a group comprising,but not limited to, perimenopausal-, post menopausal-, cancer-related-,hypogonadism-, and osteoporosis-hormonal insufficiencies.

As used herein, a “drug use” includes pharmaceutical drug use andsubstance abuse.

As used herein, a “surgery” refers to the performance of an operationincluding reconstructive, cosmetic, and restorative procedures andremoval of an organ or tissue or some portion thereof.

As used herein, a “radiation” refers to treatment by photons, electrons,neutrons or other ionizing radiation.

As used herein, a “chemotherapy” refers to the administration of anyagent that mediates the regression of malignant growth by inducing celldeath or retarding cell growth through DNA damaging and non-DNA damagingmechanisms.

As used herein, a “nerve injury” refers to damage of the somatic nerveor autonamic nerve that controls sensation and blood flow to the penis,vagina or the clitoris. The injury can occur as a result of blunt orpenetrating trauma to the pelvis, perineum or the penis as well assurgery or irradiation of the pelvic organs or the external genitalia.

As used herein, a “severe vascular insufficiency” refers to nearcomplete cessation of blood flow to the penis, vagina, or clitoris dueto damage of both internal pudendal or penile arteries.

As used herein, a “mild vascular insufficiency” refers to mild tomoderate restriction of blood flow to the penis, vagina, or clitoris andthus impairs erectile function or female sexual arousal due to partialdamage to the pudenal or penile arteries.

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections thatfollow.

B. Methods to Prevent or Treat Male Erectile Dysfunction or FemaleSexual Arousal Disorder

In one aspect, the present invention provides for a method forpreventing or treating male erectile dysfunction or female sexualarousal disorder, which method comprises administering to a mammal towhom such prevention or treatment is needed or desirable, an effectiveamount of a factor, wherein the factor is VEGF, BDNF, bFGF, NT-3, NT-4,PDGF, or Ang-1, thereby preventing or treating the male erectiledysfunction or the female sexual arousal disorder in the mammal.Preferably, the factor is a full length protein or a functionalderivative or fragment thereof, or a nucleic acid encoding the factor orfunctional derivative or fragment thereof, or an agent that enhancesproduction and/or male erection or female sexual arousal stimulatingfunction of the factor.

Any mammal can be treated with the present method. Preferably, maleerectile dysfunction or female sexual arousal disorder in humans aretreated or prevented. When human patients are treated, any suitablefactor protein, or a functional derivative or fragment thereof, or anysuitable factor nucleic acid, or a functional derivative or fragmentthereof, can be used. Preferably, the factor protein, or a functionalderivative or fragment thereof, or factor nucleic acid, or a functionalderivative or fragment thereof, is of human origin. But, suitable factorprotein, or a functional derivative or fragment thereof, or factornucleic acid, or a functional derivative or fragment thereof, ofnon-human origin can also be used when the non-human factor binds andstimulates the cell through the human factor receptor throughcross-reactivity.

VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1 proteins or functionalderivatives or fragments thereof, or a nucleic acid encoding VEGF, BDNF,bFGF, PDGF, NT-3, NT-4, Ang-1, or functional derivatives or fragmentsthereof, can be prepared by any methods known in the art, e.g.,synthetic methods, recombinant methods or a combination thereof. Anysuitable DNA construct encoding VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, orAng-1 can be used in the present invention. Such constructs include, butare not limited to VEGF-GenBank accession number M32977 (SEQ ID NO:9),BDNF-GenBank accession number M61176 (SEQ ID NO:10), bFGF-GenBankaccession number E02544 (SEQ ID NO:11), NT-3-GenBank accession numberM37763 (SEQ ID NO:12), NT-4-GenBank accession number M86528 (SEQ IDNO:13), and PDGF-GenBank accession number X02811 (SEQ ID NO:14) (PDGF-α)and X03795 (SEQ ID NO:15) (PDGF-β), and Ang-1-GenBank accession numberU83508 (SEQ ID NO:16). Further contemplated for use in the presentinvention are the DNA sequences and resultant proteins described in U.S.Pat. No. 5,607,918, U.S. Pat. No. 5,438,121, U.S. Pat. No. 5,229,500,U.S. Pat. No. 5,180,820, U.S. Pat. No. 5,387,673, U.S. Pat. No.5,155,214, U.S. Pat. No. 5,026,839, and U.S. Pat. No. 6,037,320.

Any suitable method for the production or the stabilization of the VEGF,BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1 protein known to a skilledartisan may be used in this invention. Formulations that may be used inthese inventions include, but are not limited to those described in U.S.Pat. No. 5,217,954, U.S. Pat. No. 5,235,043, U.S. Pat. No. 5,986,070,U.S. Pat. No. 6,077,829, U.S. Pat. No. 5,130,418, U.S. Pat. No.5,188,943, and U.S. Pat. No. 5,770,228.

The formulation, dosage and route of administration of theabove-described compositions, combinations, preferably in the form ofpharmaceutical compositions, can be determined according to the methodsknown in the art (see e.g., Remington: The Science and Practice ofPharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, April1997; Therapeutic Peptides and Proteins: Formulation, Processing, andDelivery Systems, Banga, 1999; and Pharmaceutical FormulationDevelopment of Peptides and Proteins, Hovgaard and Frkjr (Ed.), Taylor &Francis, Inc., 2000; Medical Applications of Liposomes, Lasic andPapahadjopoulos (Ed.), Elsevier Science, 1998; Textbook of Gene Therapy,Jain, Hogrefe & Huber Publishers, 1998; Adenoviruses: Basic Biology toGene Therapy, Vol. 15, Seth, Landes Bioscience, 1999; BiopharmaceuticalDrug Design and Development, Wu-Pong and Rojanasakul (Ed.), HumanaPress, 1999; Therapeutic Angiogenesis: From Basic Science to the Clinic,Vol. 28, Dole et al. (Ed.), Springer-Verlag New York, 1999). Thecompositions, combinations or pharmaceutical compositions can beformulated for oral, rectal, topical, inhalational, buccal (e.g.,sublingual), parenteral (e.g., subcutaneous, intramuscular, intradermal,intracavernous, or intravenous), transdermal administration or any othersuitable route of administration. The most suitable route in any givencase will depend on the nature and severity of the condition beingtreated and on the nature of the particular composition, combination orpharmaceutical composition which is being used.

Any form of male erectile or female sexual arousal disorder can betreated by the present method. The present invention provides a methodfor the treatment or prevention of male erectile dysfunction induced byor secondary to nerve dysfunction, nerve injury, arterial insufficiency,venous leakage, severe vascular insufficiency, mild vascular disease,hormonal insufficiency, drug use, surgery, chemotherapy or radiation. Inanother aspect, the present invention provides a method for thetreatment and prevention of female sexual arousal disorder induced by orsecondary to nerve dysfunction, nerve injury, arterial insufficiency,severe vascular insufficiency, mild vascular disease, hormonalinsufficiency, drug use, surgery, chemotherapy or radiation.

In a preferred embodiment, the factor protein or a functional derivativeor fragment thereof, or a nucleic acid encoding the factor or functionalderivative or fragment thereof, or an agent that enhances productionand/or female sexual arousal stimulating function of the factor, isadministered in an amount sufficient to improve blood flow andregenerate nerve and smooth muscle in the clitoris and vaginal wall. Apreferred route of administration is topically in a cream or viainjection to the clitoris and vaginal wall of the patient.

In another preferred embodiment, the present invention provides for amethod of treatment for the treatment of male erectile dysfunction orfemale sexual arousal disorder by administering the factor protein or afunctional derivative or fragment thereof, or a nucleic acid encodingthe factor or functional derivative or fragment thereof, or an agentthat enhances production and/or the male erection or female sexualarousal stimulating function of the factor, by intracavernous injection.

C. Combinations and Kits

In another aspect, the present invention provides for a combination forpreventing or treating male erectile dysfunction or female sexualarousal disorder, which combination comprises: a) an effective amount ofan agent that stimulates male erectile or female sexual function; and b)an effective amount of one or more factors, wherein the factor is a fulllength protein or a functional derivative or fragment thereof, or anucleic acid encoding said factor or functional derivative or fragmentthereof, or an agent that enhances production and/or male erection orfemale sexual arousal stimulating function of said factor and saidfactors are selected from the group consisting of vascular endothelialgrowth factor (VEGF), brain-derived growth factor (BDNF), basicfibroblast growth factor (bFGF), neurotrophin-3 (NT-3), neurotrophin-4(NT-4), platelet-derived growth factor (PDGF), and angiopoietin-1(Ang-1).

The instant invention may further comprise the coadministration of anagent that enhances, complements, or is synergistic with the erectilestimulating or female sexual arousal stimulating activity of VEGF, BDNF,bFGF, PDGF, NT-3, NT-4, or Ang-1, or some combination thereof. The agentmay comprise an agent with independent pharmacologic activity or onethat prolongs the functional or structural half-life of VEGF, BDNF,bFGF, PDGF, NT-3, NT-4, or Ang-1, or some combination thereof. Thisinvention also contemplates the administration of such VEGF, BDNF, bFGF,PDGF, NT-3, NT-4, or Ang-1, a combination thereof, with or without anaccompanying agent simultaneously or separately to maximize the maleerectile or female sexual arousal stimulating activity.

The present invention provides for administration of a combination offactors to treat or prevent male erectile dysfunction or female sexualarousal disorder. The combinations of VEGF and NT-3, VEGF and NT-4, orVEGF and BDNF are contemplated as useful combinations when thedysfunction or disorder is induced by or secondary to nerve injury. Thecombinations of VEGF and PDGF, VEGF and bFGF, and VEGF and Ang-1 arecontemplated as useful combinations when the dysfunction or disorder isinduced by or secondary to severe vascular insufficiency. Theadministration of VEGF alone is contemplated as useful when thedysfunction or disorder is induced by or secondary to mild vasculardisease.

In a further embodiment of the invention, the VEGF, BDNF, bFGF, PDGF,NT-3, NT-4, or Ang-1, or some combination thereof may be delivered tosaid mammal using a gene therapy vector. The exemplary vectors include,but are not limited to retroviruses, adenoviruses, adeno-associatedviruses, lentiviruses, herpesviruses, and vaccinia viruses vectors.Replication-defective recombinant adenoviral vectors can be produced inaccordance with known techniques. See, Quantin, et al., Proc. Natl. AcadSci. USA, 89:2581-2584 (1992); Stratford-Perricadet, et al., J. Clin.Invest., 90:626-630 (1992); and Rosenfeld, et al., Cell, 68:143-155(1992).

The vector may include an expression construct of VEGF, BDNF, bFGF,PDGF, NT-3, NT-4, or Ang-1 nucleic acid, a functional derivativefragment thereof under the transcription control of a promoter. In apreferred embodiment, the gene therapy vector may be administered byintracavernous injection of about 0.5 to 2 ml of AAV-VEGF, AAV-BDNF,AAV-bFGF, AAV-PDGF, AAV-NT-3, AAV-NT-4, or AAV-Ang-1 at a concentrationof about 10¹⁰ virus titer. An example of a suitable promoter that may beused is the 763-base-pair cytomegalovirus (CMV) promoter. Viralpromoters, cellular promoters/enhancers and induciblepromoters/enhancers that could be used in combination with the nucleicacid contemplated for use in this invention include those listed in Jin,et al., U.S. Pat. No. 6,251,871. The expression construct may beinserted into a vector, such as pUC118, pBR322, or other known plasmidvectors, that includes an origin of replication. See, for example,Current Protocols in Molecular Biology, Ausubel, et al. eds., John Wiley& Sons, Inc. (2000), Sambrook, et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory press, (1989). The plasmid vectormay also include a selectable marker such as the β-lactamase gene forampicillin resistance, provided that the marker polypeptide does notadversely effect the metabolism of the organism being treated.

The preferred embodiment of the VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, orAng-1, or agent nucleic acid may be delivered via an adeno-associatedviral (AAV) vector. These viruses are single-stranded DNA, nonautotomousparvoviruses that are able to integrate efficiently into the genome ofnondividing cells of a very broad host range. Although ubiquitous innature, AAV has not been shown to be associated with any known humandisease and does not elicit an immune response in an infected humanhost. GOODMAN & GILMAN, PHARMACOLOGICAL BASIS OF THERAPEUTICS, 9th ed.,McGraw-Hill Press (1996), p. 77-101. The method to produce purifiedreplication deficient recombinant adeno-associated virions is describedin Dwarki, et al., U.S. Pat. No. 6,221,646 B1, and its contents areincorporated in their entirety herein.

The present invention contemplates the use of AAV vectors that are knownto those of skill in the art. For example, the vectors and vectorproduction methods described in U.S. Pat. Nos. 5,589,377; 5,753,500; and5,693,531. In another embodiment of this invention, the nucleic acid maybe delivered in a retroviral vector, using vector and production methodknown in the art. See, for example, U.S. Pat. No. 5,830,725. A furtherembodiment would deliver the nucleic acid in an adenovirus vector, usingvectors and vector production methods known in the art. See, forexample, U.S. Pat. Nos. 6,063,622; 6,083,750; 5,994,128; and 5,981,225.

In a further embodiment of the invention, factors used in the presentmethod and the encoding nucleic acids may be delivered in a liposome.Liposomes are vesicular structures characterized by a phospholipidbilayer membrane and an inner aqueous medium. Multilamellar liposomeshave multiple lipid layers separated by aqueous medium. They formspontaneously when phospholipids are suspended in an excess of aqueoussolution. The lipid components undergo self-rearrangement before theformation of closed structures and entrap water and dissolved solutesbetween the lipid bilayers (Ghosh and Bachhawat, 1991). For a review ofthe procedures for liposome preparation, targeting and delivery ofcontents, see Mannino and Gould-Fogerite, Bio Techniques, 6:682 (1988).See also, Current Protocols in Molecular Biology, Ausubel, et al. eds.,John Wiley & Sons Press (2000), Chapters 9 and 16. The process of makingand loading the liposomes with nucleic acid or protein may employtechniques known in the art. See, for example, U.S. Pat. Nos.,6,007,838; 6,197,333 B1; 6,133,026; 6,120,798; 5,939,096; 5,662,931;5,552,157; and 5,270,053.

According to the present invention, the VEGF, BDNF, bFGF, PDGF, NT-3,NT-4, or Ang-1 peptides, proteins, polynucleotides, nucleic acids, oragent that enhances production and/or erection or sexual arousalstimulating function of said factor may be formulated for intracavernousinjection, subcutaneous injection, intravenous injection, intramuscularinjection, intradermal injection, or topical administration. The methodmay employ formulations for injectable administration in unit dosageform, in ampules or in multidose containers, with an added preservative.The formulations may take such forms as suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, sterile pyrogen-free water orother solvents, before use. Topical administration in the presentinvention may employ the use of a foam, gel, cream, ointment,transdermal patch, or paste.

Pharmaceutically acceptable compositions and methods for theiradministration that may be employ for use in this invention include, butare not limited to those described in U.S. Pat. Nos. 5,736,154;6,197,801 B1; 5,741,511; 5,886,039; 5,941,868; 6,258,374 B1; and5,686,102.

One preferred embodiment is the intracavernous administration of about0.5 to 2.0 ml of the AAV-VEGF, AAV-BDNF, AAV-bFGF, AAV-PDGF, AAV-NT-3,AAV-NT-4, or AAV-Ang-1 gene therapy vector at a concentration of about10¹⁰ virus titer.

The magnitude of a therapeutic dose in the acute or chronic treatment oferectile dysfunction or female sexual arousal disorder will vary withthe severity of the condition to be treated and the route ofadministration. The dose, and perhaps dose frequency, will also varyaccording to age, body weight, condition and response of the individualpatient. A preferred dosage for the treatment or prevention of maleerectile dysfunction and/or female sexual arousal disorder is about10-200 mcg/70 Kg body about once every two to six months.

One preferred embodiment of the present invention contemplates theadministration by the application of a cream or by an injection to theclitoris and vaginal wall of a patient with female sexual arousaldisorder in an amount sufficient to improve blood flow and regeneratenerve and smooth muscle in the clitoris and vaginal wall.

It should be noted that the attending physician would know how to andwhen to terminate, interrupt or adjust therapy to lower dosage due totoxicity, or adverse effects. Conversely, the physician would also knowhow to and when to adjust treatment to higher levels if the clinicalresponse is not adequate (precluding toxic side effects).

Any suitable route of administration may be used. Dosage forms includetablets, troches, cachet, dispersions, suspensions, solutions, capsules,patches, and the like. See, Remington's Pharmaceutical Sciences.

In practical use, VEGF, BDNF, bFGF, PDGF, NT-3, NT-4, or Ang-1 peptides,proteins, polynucleotides, nucleic acids, or agent that enhancesproduction and/or erection or sexual arousal stimulating function ofsaid factor may be combined as the active in intimate admixture with apharmaceutical carrier or incipient according to conventionalpharmaceutical compounding techniques. The carrier may take a wide formof preparation desired for administration, topical or parenteral. Inpreparing compositions for parenteral dosage form, such as intravenousinjection or infusion, similar pharmaceutical media may be employed,water, glycols, oils, buffers, sugar, preservatives, liposomes, and thelike known to those of skill in the art. Examples of such parenteralcompositions include, but are not limited to dextrose 5% w/v, normalsaline or other solutions. The total dose of VEGF, BDNF, bFGF, PDGF,NT-3, NT-4, or Ang-1 to be administered may be administered in a vial ofintravenous fluid, ranging from about 1 ml to 2000 ml. The volume ofdilution fluid will vary according to the total dose administered.

The invention also provides for kits for carrying out the therapeuticregimens of the invention. Such kits comprise in one or more containerstherapeutically effective amounts of the VEGF, BDNF, bFGF, PDGF, NT-3,NT-4, Ang-1, or an agent stimulating the production and/or function ofVEGF, BDNF, bFGF, PDGF, NT-3, NT-4, Ang-1, or some combination thereofin pharmaceutically acceptable form. Preferred pharmaceutical formswould be in combination with sterile saline, dextrose solution, orbuffered solution, or other pharmaceutically acceptable sterile fluid.Alternatively, the composition may be lyophilized or dessicated; in thisinstance, the kit optionally further comprises in a container apharmaceutically acceptable solution, preferably sterile, toreconstitute the complex to form a solution for injection purposes.Exemplary pharmaceutically acceptable solutions are saline and dextrosesolution.

In another embodiment, a kit of the invention further comprises a needleor syringe, preferably packaged in sterile form, for injecting thecomposition, and/or a packaged alcohol pad. Instructions are optionallyincluded for administration of composition by a physician or by thepatient.

In yet another embodiment, a kit of the invention further comprises in acontainer a pharmaceutically acceptable composition suitable for topicaladministration. Instructions are optionally included for administrationof composition by a physician or by the patient.

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLE 1 The Effect of Vascular Endothelial Growth Factor (VEGF) on aRat Model of Traumatic Arteriogenic Erectile Dysfunction

Animal Model

Fifty 3-months-old male Sprague Dowley rats weighing 350˜400 grams wereanesthetized with intraperitoneal pentobarbital (35 mg/kg) after beingsedated with inhalation of methoxyflurane. Midline laparotomy wasperformed to identify the iliac vessels. Under operating microscope, theiliac veins were carefully dissected to expose the internal iliacarteries that vary from 2 to 4 in number. Rats in the experimental group(n=44) underwent bilateral ligation of internal iliac arteries at theirorigin. A separate incision was made in the perineum to identify one ofthe crura. A 23-gauge scalp vein needle was inserted to the crus andconnected to a pressure monitor for intracavernous pressure monitoringduring electrostimulation of the cavernous nerve. Additional ligation ofarterial branches was performed until minimal or no intracavernouspressure rise during electrostimulation. Intracavernous injection of 4μg of VEGF in PBS (phosphate-buffered saline) with 0.1% BSA (bovineserum albumin); 2 μg of VEGF in PBS with 0.1% BSA; or PBS solution with0.1% BSA was then administered through the same needle to 3 groups ofrats (n=16, 12 and 16 respectively). The wound was closed in layers andthe animals were closely monitored for up to 6 weeks. At week 1, 2 and 6roughly one-third of rats from each group underwent electrostimulationof the cavernous nerve to assess erectile function and then sacrificed.Penile tissues of 3 rats randomly chosen from each of the threesubgroups at 2 and 6 weeks were obtained for immunohistochemicalstaining and electron microscopic examination.

Electrical Stimulation

Bipolar platinum-wire electrodes were used to stimulate the cavernousnerve. The exposed end of the electrodes were hooked around the nerve tobe stimulated, with the positive electrode being positioned proximallyand the negative electrode two to three mm distally. Stimulus parameterswere 1.5 volts, frequency of 20 pulses per second, pulse width of 0.2msec, and the duration of 50 seconds. Intracavernous pressure wasmonitored and recorded by inserting a number 23 scalp vein needle to oneof the crura and connected to a pressure monitor.

Immunohistochemical Staining

Penile tissue were fixed for 3 hours in a cold, freshly preparedsolution of 2% formaldehyde, 0.002% picric acid in 0.1 M. phosphatebuffer, pH 8.0. Tissues were cryoprotected for 24 hours in cold 30%sucrose in 0.1 M. phosphate buffer, pH 8.0. They were then embedded inO.C.T. compound (Tissue-Tek, Miles Laboratory), frozen in liquidnitrogen, and stored at −70° C. After freezing, Cryostat tissue sectionswere cut at 10 μm., adhered to charged slides, air-dried, and hydratedfor 5 min. with 0.05 M. sodium phosphate buffer (PBS, pH 7.4). Sectionswere treated with hydrogen peroxide/methanol to quench endogenousperoxidase activity. After rinsing with water, sections were washedtwice in PBS for 5 min. followed by 30 min. of room-temperatureincubation with 3% horse serum/PBS/0.3% triton X-100. After drainingsolution from sections, tissues were incubated for 60 min. at roomtemperature with mouse monoclonal anti-nNOS (Transduction Laboratories,Lexington, Ky.) at a 1:500 dilution. After washing for 5 min. withPBS/TX and then for 5 min. twice with PBS alone, sections wereimmunostained with the avidin-biotin-peroxidase method (Elite ABC,Vector Labs, Burlingame Calif.), with diaminobenzidine as the chromagen,followed by counterstaining with hematoxylin.

Electron Microscopy

The penis was dissected, thinly sliced (˜1 mm thick) and placed inKarnovsky's fixative (1% para-formaldehyde/3% glutaraldehyde/0.1 Msodium cacodylate buffer, pH 7.4) at room temperature for 30 minutes andthen stored at 4° C. The fixed tissue was then rinsed in buffer,post-fixed in 2% aqueous OsO4, and stained en bloc with uranyl acetatebefore being dehydrated in ethanol, cleared in propyline oxide, andembedded in eponate 12 (Ted Pella Co., Redding, Calif.). Thick sectionswere cut and stained with toludine blue, examined under light microscopeto select the area to be thin-sectioned. Thin sections were cut by Leicaultracut E microtome (Bannockburn, Ill.), stained with uranyl acetateand Reynold's Lead to enhance contrast and examined under Philips Tecnai10 electron microscope (Eidhoven, Netherlands).

Statistics

Data were evaluated with Mann-Whitney rank-sum test. Significance wasdefined as p<0.05.

Erectile Function

The peak sustained intracavernous pressure during electrostimulation ofthe cavernous nerve is shown in Table I. There is no difference inintracavernous pressure between the sham operated and the normal rats.After bilateral ligation of the internal iliac arteries, theintracavernous pressure immediately dropped to around 20 cm H₂O andproduced no or minimal pressure increase in response to neurostimulationin all rats. In the PBS-treated group, poor erectile response persistedat weeks 1 and 2 and slight recovery of erectile function was noted atweek 6.

In the VEGF-treated rats, at weeks 1 and 2, moderate recovery oferectile function was noted in the 4-μg group but not the 2-μg group. Atweek 6, statistically significant improvement in intracavernous pressurewas seen in both the 2-μg and 4-μg groups as compared with thePBS-treated group. The intracavernous pressure of the 4-μg group wasalso significantly higher than that of the 2-μg group.

To identify the new source of blood flow in the 4-μg VEGF-treated group,we noted a decrease in erectile response after clamping one externaliliac artery and no erectile response at all after clamping bothexternal iliac arteries. This strongly suggests that the collateralvessels were derived from the external iliac arteries. TABLE 1 Peaksustained intracavernous pressure (cm H₂0) during electrostimulation ofthe cavernous nerves in saline- and VEGF-treated rats Saline treatedGroup VEGF-treated Group PBS + 0.1% BSA PBS + 2 μg VEGF PBS + 4 μg VEGFWeek 1 20.33 ± 3.45  23.50 ± 2.38  71.17 ± 16.89 (n = 6) (n = 4) (n = 6)Week 2 27.75 ± 9.70  43.00 ± 8.37  86.25 ± 8.18  (n = 4) (n = 4) (n = 4)Week 6 46.75 ± 14.85 69.00 ± 8.83  96.67 ± 13.50 (n = 6) (n = 4) (n = 6)Sham operated group (n = 6): 98 ± 8.50Histochemistry

There was a trend of decreased nNOS-immunoreactive in both dorsal andintracavernous nerves two weeks after arterial ligation in allsubgroups. At week 6, moderate recovery of nNOS-positive nerve fiberswas noted in both dorsal and intracavernous nerves in both 2 & 4 μgVEGF-treated groups but not in the PBS-treated group. However, thedifferences were not statistically significant by computer-assistedimage analysis.

Electron Microscopy

Dorsal Nerve:

In sham-operated rats, no difference was noted between specimensobtained from the 2 and 6-week groups. The dorsal nerve in these ratswas filled with both myelinated and non-myelinated nerve bundles. Themean diameter of the individual myelinated nerve axon was 4.42±1.36 μm(excluding myelin sheath). The mean thickness of the myelin sheath was0.58±0.21 μm. The mean diameter of the non-myelinated nerve fibers was0.96±0.37 μm. The nuclei of Schwann cells were seen occasionally nearthe nerve fibers.

In ligated+PBS treated rats, dramatic changes were noted at week 2.There was an increase in the number of Schwann cells and many of whichcontained vacuous within the cytoplasm. There was also a decrease in thenumber of both non-myelinated and myelinated nerve fibers. Overall thesize of the axons was smaller than that of the controls, and many of thenon-myelinated nerve fibers were smaller and less discrete. At week 6,varying degree of regeneration of both myelinated and non-myelinatednerve fibers was apparent. However, the nerve fibers of both themyelinated and non-myelinated nerve fibers were still smaller than thecontrol groups. (mean myelinated axon 3.17±1.01 μm, myelin sheath0.46±0.11 μm, non-myelinated axon 0.81±0.38 μm, p=0.062, 0.189, and0.069 respectively compared with those of the sham group.) Many of thenon-myelinated fibers were less than one third of the size of the largerones. There was also an increase in the number of nucleated Schwanncells.

At week 6, in ligated rats treated with 4 μg of VEGF, the mean diameterof the myelinated axons (6.19±2.38 μm) was larger than the ligated+PBStreated ones (3.17±1.01 μm) with a p=0.008. The mean diameter of thenonmyelinated axons was 0.82±0.45 μm which was similar to that of theligated+PBS treated rats (0.81±0.38 μm). However, the non-myelinatednerve fibers appeared more even in size.

Intracavernosal Smooth Muscle:

In sham-operated rats, no difference was noted between the specimensfrom the 2 and 6-week groups. The smooth muscle cells, most of whichwere arranged in clusters, were embedded in fine strands offibro-connective tissue. The cytoplasm of these myocytes containedabundant contractile myofilaments and dense bodies. Occasionally, smallaggregates of organelles, including mitochondria, rough endoplasmicreticulum and Golgi apparatus, were found adjacent to the nucleus. Thecell membrane (sarcolemma) was consisted of many alternating dense andlight bands with the latter containing numerous pinocytotic vesicles(caveolae). The intercellular spaces were narrow and many cell-cellcontacts (gap junctions) were visible. Nerve terminal varicosities wereseen occasionally near clusters of smooth muscle cells.

At week 2, in ligated+PBS-treated rats, many atrophic smooth musclecells separated by large amounts of collagen fibers were noted. Theseatrophic smooth muscle cells appeared scattered in a sheet of connectivetissues while normal smooth muscle cells were separated only by minimalamounts of connective tissues. At week 6, the number of normal-appearingsmooth muscle cells increased, although many of them still showedsignificant loss of myofilaments.

In ligated+4 μg of VEGF-treated rats (both 2 and 6-week groups), most ofthe smooth muscle cells appeared normal with large amount ofmyofilaments and narrow intercellular spaces.

Endothelium:

In sham-operated rats, the cavernous sinusoids were lined with intactendothelium, the cytoplasm of which contained numerous pinocytoticvesicles (caveolae), mitochondria, rough endoplasmic reticulum, andGolgi apparatus. The nuclei of the endothelial cells were sparsely seenand appeared flattened. In ligated+PBS-treated rats, (both 2 and 6-weekgroups) the capillary and cavernous sinusoidal endothelium appearednormal, although increase in the number of endothelial cells could beseen in some fields. In ligated+4 μg of VEGF-treated rats, (both 2 and6-week groups) striking differences were noted when compared with othergroups of rats. Many reactive endothelial cells with plump nuclei couldbe seen lining the sinusoids and capillaries. These endothelial cellswere larger and more numerous indicative of endothelial cell hypertrophyand hyperplasia. Lee M C, et al., J. Urol. 167:761-7 (2002).

References

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EXAMPLE 2 Intracavernosal Vascular Endothelial Growth Factor (VEGF) andAdeno-Associated Virus Mediated VEGF Gene Therapy Prevents and ReversesVenogenic Erectile Dysfunction in Rats

Animal Groups

Male Sprague-Dawley rats age 3-6 months (wt 350-450 grams) were used inthis study. They were housed in our animal care facility with rat chowand water available ad libitum on a 12 hr light/dark cycle. All animalcare, treatments and procedures were performed in compliance withrequirements of the Committee on Animal Research at our institution.Rats were randomly divided for the animal model of vasculogenic ED(Experiment 1) and the VEGF prevention trial (Experiment 2). For theVEGF treatment trial (Experiment 3), the animals underwent castrationand then were treated with VEGF after venous leak was demonstrated, toevaluate the efficacy of VEGF treatment in reversing establishedvenogenic ED.

Experiment 1:

To determine normal values for rodent pharmacologic cavemosometry andvalidate the model of venogenic ED in the rat, vasculogenic ED wasinduced. Arterial insufficiency was produced after performing abilateral ligation of the internal iliac arteries. The acute and chroniceffects of arterial insufficiency were evaluated 7 days and 30 daysafter bilateral iliac artery ligation (n=7). Venogenic ED was induced bycastration, and pharmacologic cavernosometry was performed 6 weeks aftersurgery (n=10). Control animals underwent a sham laparotomy and studied6 weeks later (n=13).

Experiment 2:

For the prevention trial of VEGF in rats with venogenic erectile ED,adult males were castrated and immediately treated with hormone,intracavernosal VEGF. Hormone replacement was accomplished using asubcutaneously placed testosterone-filled silastic implant (n=6), aspreviously described.¹⁷ A therapeutic testosterone serum titer wasconfirmed in this animal group by testosterone radioimunoassay performedby the biomedical core lab at our institution.¹⁸ Intracavernoustreatment with VEGF was administered using either recombinant VEGFprotein (n=5) or an adeno-associated virus vector expressing the VEGFgene (AAV-VEGF, n=14). Control animals received an silastic implantcontaining saline (n=3), an intracavernous injection of normal saline(n=3) or an adenovirus transfection vector expressing lacZ reporter genewithout the VEGF gene (AAV-LacZ, n=11).

Experiment 3:

The trial of VEGF treatment was performed in castrated animals that wereshown, 6-8 weeks following castration, to develop venogenic erectile EDby pharmacologic cavernosometry. These animals (n=8) were treated withintracavernous VEGF protein and then after one month cavernosometryrepeated to measure the effect of VEGF treatment.

Animal Treatments

Surgical Preparation:

Prior to all surgical procedures, animals received anesthesia consistingof isoflurane inhalation as pre-anesthetic followed by anintraperitoneal injection of sodium pentobarbital (40 mg/kg). After theanimal was asleep, electric clippers were used to trim the ventralabdominal hair and the skin was prepped with clorhexidine scrub.Antiseptic technique was maintained for all procedures. Followingsurgery, the anterior abdominal fascia and skin were approximated with4-0 silk suture, analgesic buprenorphine (0.5 mg/kg SC) was administeredand the animal allowed to awaken covered with a heating pad. Euthanasiawas accomplished by an intraperitoneal injection of sodium pentobarbital(200 mg/kg) followed by bilateral thoracotomy when the animal was fullyasleep.

Arterial Ligation Surgery:

A 2 cm midline longitudinal low abdominal incision was made and awheatlander retractor placed so that the plane between the prostate andsigmoid colon could be bluntly opened. A dissecting microscope with2.5-10× objectives was essential to safely performing this procedure.Using sterile cotton-tipped swabs, the iliac artery bifurcation wasidentified and the common iliac exposed to the external iliac take-off.The internal iliac arteries were identified as those medial branches offthe common iliac between the iliac bifurcation and the take-off of theexternal iliac. These were doubly ligated using 7-0 nylon sutures. Afterthis was performed on both the right and left side, the incision wasclosed and the animal recovered as noted above.

Castration:

A 2 cm midline longitudinal low abdominal incision was made and eachtesticle was grasped using forceps and brought into the incision. Eachgubernaculum was divided using electrocautery and then the spermaticcords ligated with 4-0 silk suture and divided. After confirminghemostasis, the abdomen was closed and the rat recovered as above.

Intracorporal Injections:

A 1.5 cm oblique incision was made in the lower abdominal skin extendingfrom the midline just above the penile hilum to below the level of theglans about 1 cm lateral to the midline. The skin was sharply dissectedfrom the anterior surface of the penis and then the penis was retractedanteriorly using a towel clamp placed around it atraumatically with theforeskin left intact. Using blunt dissection the penile base and crurawere exposed. The ischiocavernosus muscles were sharply dissected offthe anterior surface of the crus until the white of the tunica albugineaof the corpora cavernosa was identified. The crus was then gentlycannulated using a 23 gage butterfly needle, and saline flush with avisual erectile response was used to confirm that the needle tip wastruly intracavernosal. The intracavernosal injections were thenadministered with either VEGF protein (Calbiochem, La Jolla, Calif.#676472) at the dosage 4 ug/injection (in 0.1 cc PBS with 0.1% BSA),AAV-VEGF (10¹⁰ viral particles in 0.1 cc NS), AAV-LacZ (10¹⁰ viralparticles in 0.1 cc NS), or 0.1 ml NS alone. The AAV-VEGF and AAV-LacZconstructs were a generous gift from Dr. Yuet W. Kan (Howard HughesMedical Research Institute, San Francisco, Calif.).^(19,20) Followinginjection, the needle was left in place for 5 minutes and then removedto allow the medication to diffuse throughout the cavernosal space.Immediately thereafter, pinpoint electrocautery was applied to theneedle hole for hemostasis and then the wound was closed and the animalrecovered as above.

Testosterone Replacement:

Following castration, testosterone- or saline-filled silastic implantswere placed in the subcutaneous tissue of the anterior abdominal wall,as previously described.¹⁷ Implants were prepared using sterile silastictubing (Dow Coming, Midland, Mich. #602-265, inner diameter 0.062″) thatwas filled with testosterone propionate powder (Sigma Chemical, St.Louis, Mo.) with the aid of wall suction. By radioimmunoassay,¹⁸ serumtestosterone titer was found to be undetectable in the castrated animalsand in the normal range for animals given testosterone implants.

Pharmacologic Cavernosometry in the Rat:

To perform pharmacologic cavemosometry, both the right and left crurawere separately cannulated using 23 gauge butterfly needles as describedabove. One cannula was flushed with sterile heparinized saline (100 Uheparin/ml NS) and attached to a pressure detector for continuousintracorporal pressure (ICP) monitoring as previously described.¹¹ Thecontralateral cannula was attached to an infusion pump (Harvard Pump,Southwick, Mass. #55-2222), filled with sterile dilute heparinizedsaline (20 U heparin/ml NS). The baseline ICP was recorded (flaccid ICP)and then a dose of papavarine (1 mg in 0.1 cc NS) was administeredthrough the infusion cannula. Five minutes was allowed for thepapavarine to diffuse throughout the corpora and then the infusioncannula was flushed with heparinized saline and the pressure monitorcannula vented to normalize ICP after flushing. After 5 minutes more,the ICP was again recorded (ICP after papavarine) and the infusionstarted. An infusion rate of 0.05 ml/min was started and increased (by0.05 ml/min every 10 seconds) until the ICP started to rise. Subsequentincreases in inflow rate were made only after the ICP reached a plateaupressure. By slowly adjusting the inflow rate, an intracorporal pressureof 100 cm H₂O (erectile pressure) was reached and the infusion raterequired to maintain this pressure recorded (the maintenance rate).After this pressure was steady for 20 seconds, the infusion wasterminated and the change in ICP over the subsequent 60 seconds wasrecorded (the drop rate).

Tissue Preparation:

After pharmacologic cavemosometry was performed, the penis was amputatedat the crural bony attachments and immediately placed in ice-coldsaline. The Y-shaped crura was sharply cut from the penile base and thena 1 mm thick slice cut for electron microscopy and placed in Karnofsky'ssolution (3% gluteraldehyde, 1% para-formaldehyde, 0.1 M sodiumcacodylate buffer, pH 7.4). A 3 mm thick section of the distal penileshaft was then cut and placed in 10% normal buffered formalin forparaffin sections, and the balance of the penile shaft was flash frozenusing dry ice in OCT compound (Sakura Finetek USA, Torrance, Calif.) forfrozen sectioning and immunohistochemistry.

Immunohistochemistry:

Frozen sections were cut at 10 microns, adhered to charged slides, airdried for 15 minutes then rehydrated with 0.05M PBS for 5 minutes.Sections were treated with hydrogen peroxide/methanol to quenchendogenous peroxidase activity. After rinsing, sections were washedtwice in PBS for 5 minutes then incubated with 3% horse serum and 0.3%triton X-100 at room temperature for 30 minutes. The serum solution wasdrained and then sections were incubated for 60 minutes with mousemonoclonal anti-alpha-smooth muscle actin (Sigma, St. Louis, Mo.) at adilution of 1:4000 in PBS. After washing, sections were immunostainedusing the avidin-biotin-peroxidase method (Elite ABC—Vector Labs,Burlingame, Calif.), with diaminobenzidine as the chromogen, followed bycounterstaining with hematoxylin. Immunochemistry was performed inpenile tissues from 4 rats randomly chosen from each subgroup.

Enzyme-Linked Immuno-Sorbent Assay:

Serum samples from both systemic and penile blood were collected afterwhole-blood centrifugation. Solid phase enzyme-linked immuno-sorbentassay for VEGF was performed using the Quantikine M mouse VEGFImmunoassay Kit (R&D Systems, Minneapolis, Minn.) as previouslydescribed.²¹ Briefly, samples were diluted and added to micro platestrip wells that were then treated with the enzyme-labeledimmunoreactant VEGF conjugate. After incubation for 2 hours and washing,the substrate solution was added and incubated for 30 minutes. The stopsolution was added and then the optical density in each well determinedusing a micro plate reader set to 450 nm. Sample results were plotted ona curve generated by the optical density of standard samples ranging inconcentration (0-500 pg/ml VEGF).

Transmission Electron Microscopy:

After fixing in Karnofsky's solution for 30 minutes at room temperature,1 mm sections at the penile base were stored at 40 C until processed, aspreviously described.²² Briefly, samples were rinsed in PBS, post-fixedin 2% aqueous OsO4 and stained en bloc with uranyl acetate. They werethen dehydrated in ethanol, cleared in propyline oxide and embedded inEponate 12 (Ted Pella Co., Redding, Calif.). Thick sections were cut andstained with toluidine blue to select specific areas for thinsectioning. Thin sections were cut, stained with both uranyl acetate andReynold's Lead, and examined under the Phillips Tecnai 10 transmissionelectron microscope. Penile tissues from 4 randomly chosen rats in eachsubgroups were subjected to electron microscopic examinations.

Statistical Analysis:

Data in the present studies was analyzed using the student's t-test(homoscedastic, 2-tailed) when 2 means were compared (Experiment 1 and2). A paired, 2-tailed t-test was used where values represent findingsbefore and after treatment in the same animals (Experiment 3).

Results

Experiment 1

Model Validation:

The first goal was to determine normal values for pharmacologiccavemosometry in a rat model of vasculogenic ED. As shown in Table 2,flaccid ICP were comparable, in the range of 30 cm H2O. After papavarineinjection, however control animals had a steep rise in ICP to >100 cmH₂O while the castrated and ligated animals had less response. Only aminimal increase in ICP (5-10 cm H₂O) was noted in the animals followingeither castration or chronic internal iliac ligation, characteristic ofvasculogenic ED. Acute ligation animals had a better response topapavarine yet significantly less than seen in normal animals. After theinfusion was started, both the control and acute ligation group promptlyachieved erectile pressure with minimal inflow required. When theinfusion was stopped, these animals had a minimal pressure drop,evidencing their intact veno-occlusive mechanism. The castration andchronic ligation groups, on the other hand, required a significantlyhigher infusion rate to maintain erectile pressure and experienced asteep pressure drop when the infusion was terminated. These findings arecharacteristic of venous leakage in the chronic ligation and castrationgroups. TABLE 2 (Experiment 1) Cavernosometric findings in rat model ofvasculogenic ED. ICP After Drop Rate in Flaccid ICP PapavarineMaintenance 1 min. (cm H₂O) (cm H₂O)* Rate (ml/min)* (cm H₂O)* Control35.4(+/−9.3)  104(+/−59) 0.024(+/−0.3)   9(+/−13) Castration22.0(+/−5.2) 35.0(+/−5.0)  1.14(+/−0.5)   75(+/−5.4) Acute 28.7(+/−7.6)73.3(+/−10)  0.06(+/−0.12) 13.2(+/−13.8) Ligation Chronic 29.3(+/−7.6)38.3(+/−19)  1.9(+/−1.8) 45.8(+/−19) Ligation

Cavernosometry was performed 6 weeks after castration, 7 days afterinternal iliac ligation in the acute ligation group, and 30 days afterligation in the chronic ligation group. (*p<0.05, for comparison ofcastration vs. control groups, and castration vs. acute ligation groups)

Experiment 2

Prevention Trial:

Our second goal was to perform a prevention trial using intracavemosalVEGF either in the form of recombinant protein or virus-directed geneexpression vector in an attempt to prevent the development of venogenicerectile ED in castrated animals. As shown in Table 3, flaccid ICP wasagain in the range of 30 cm H₂O in each of the animal groups. Afterpapavarine administration, however, both control groups (castration onlyand castration with LacZ injection) exhibited only a weak rise in ICP(5-10 cm H₂O), required a significant infusion rate to sustain anerectile ICP of 100 cm H₂O, and had a steep pressure drop after theinflow was terminated. In contrast, the 3 treatment groups exhibitednearly normal erectile function with high ICP in response to papavarine,a very low maintenance rate to sustain erectile ICP and minimal pressuredrop when the infusion was stopped. Of note, the VEGF gene-treatedanimals showed marginally less erectile function with a lesser responseto papavarine and a higher drop rate than the animals treated witheither testosterone replacement or intracavemosal VEGF protein. TABLE 3(Experiment 2) Cavernosometric findings in castrated animals afterprevention trial of testosterone replacement (C + Testosterone), VEGFprotein treatment (C + VEGF), AAV-VEGF gene therapy (C + VEGF gene), orLacZ control (C + LacZ control). Flaccid ICP After Maintenance Drop Ratein ICP Papavarine Rate 1 min. (cm H₂O) (cm H₂O)* (ml/min)* (cm H₂O)* C +saline 23.4 29.4(+/−15) 0.51(+/−0.26) 56.1(±/−15) (+/−5.3) C + 28.987.7(+/−26) 0.09(+/−0.1) 11.7(+/−16) Testosterone (+/−7.5) C + VEGF 27.885.0(+/−28) 0.04(+/−0.09)  9.0(+/−20) (+/−5.2) C + 23.4 61.4(+/−36)0.04(+/−0.03) 27.8(+/−18) VEGF gene (+/−7.1) C + LacZ 27.0 36.0(+/−12.8)0.25(+/−0.31) 58.6(+/−8.1) (+/−6.7)

Pharmacologic cavernosometry was performed 9 weeks after castration.(*p<0.05, comparing castration only to the treatment groups, excludingthe LacZ control group.)

Experiment 3

Treatment Trial:

The third phase was to perform a treatment trial using intracavemosalVEGF in animals with venous leak. The goal was to assess the efficacy ofVEGF at reversing established venogenic erectile dysfunction in ananimal model. Animals were castrated and then 4-6 weeks later underwentcavemosometry. As shown in Table 4, before VEGF treatment, this animalgroup displayed a weak response to papavarine with intracorporalpressure reaching 33 cm H₂O compared to normal animals who attain nearly100 cm H₂O with such treatment (see Table 2). Also, these castratesrequired a relatively high maintenance rate (0.19 ml/min.) to achieveerectile pressure and a steep drop rate when the infusion was terminated(45.1 cm H₂O in 60 seconds), evidencing venous leak. After these animalsreceived intracorporal VEGF treatment, however, nearly normal erectilefunction returned with a prompt rise in intracorporal pressure afterpapavarine (to 84 cm H₂O), a low maintenance rate (0.08 ml/min.) toachieve erectile pressure and a minimal drop in intracorporal pressure(17.4 cm H₂O in 60 seconds) after the infusion was terminated. TABLE 4(Experiment 3) Cavernosometric findings in VEGF treatment trial. FlaccidICP After Drop Rate in ICP Papavarine Maintenance 1 min. (cm H₂O) (cmH₂O)* Rate (ml/min)* (cm H₂O)* Before VEGF 22.4 33.0 0.19(+/−0.18)45.1(+/−18) treatment (+/−6.9) (+/−12.3) (6 wks after castration) 1month 25.3 83.9 0.08(+/−0.15) 17.4(+/−24) following (+/−8.5) (+/−31)VEGF treatment

Animals were castrated and then shown to have venous leak (afterapproximately 6 weeks) by pharmacologic cavemosometry. They were thentreated with intracavemosal VEGF and one month later underwent repeatcavemosometry. (*p<0.05)

Immunohistochemistry:

Cross-sectional micrographs of the rat penis at the proximal shaft wereexamined after immunohistochemistry for alpha actin. Alpha actin, amarker for penile smooth muscle, stains brown and can be seensurrounding the sinusoidal spaces. Qualitatively, we see decreasedsmooth muscle content 6 weeks after castration compared to the normalcontrol. In castrates treated with either testosterone replacement orintracavemosal VEGF protein, the quantity of smooth muscle returns tonormal morphology. Computerized image analysis with Adobe Photoshop wasused to quantify the area of immunostaining by counting the number ofdigitized pixels corresponding to the area of brown staining, therebyproviding some numerical comparison of the quantity of smooth muscle ineach specimen. This analysis shows the following pixel count: sham(43518), castrated (37214), AAV-VEGF treated (51690), VEGF proteintreated (52990).

Transmission Electron Microscopy

Dorsal Nerve:

In sham-operated rats, the dorsal nerve was filled with both myelinatedand non-myelinated nerve bundles. The mean diameter of the individualmyelinated axon (excluding myelin sheath) was 2.54±1.04 μm. The meanthickness of the myelin sheath was 0.74±0.21 μm. The mean diameter ofthe non-myelinated axon was 0.97±0.35 μm. The cytoplasm and nuclei ofSchwann cells were seen occasionally near the nerve fibers.

In castrated rats, with or without LacZ injection, the diameter of boththe myelinated and non-myelinated axons appeared smaller than those ofthe sham-operated rats. Mean diameters were the following: myelinatedaxon 1.64±1.0 μm; myelin sheath 0.49±0.13 μm; non-myelinated axon0.64±0.32 μm. Comparing the castrated rats to the sham-operated rats,the p values were 0.06, 0.004 and 0.001 respectively. Manynon-myelinated nerve fibers became indistinct and smaller. There wasalso an increase in the number of nucleated Schwann cells.

Although many small myelinated nerve fibers were still present incastrated rats treated with VEGF or AAV-VEGF, larger fibers with thickmyelin sheaths were also noted. The mean diameter of the myelinatednerve and myelin sheath were 2.36±0.92 μm and 0.93±0.44 μm respectively.The non-myelinated nerve fibers were more clearly defined but were notas abundant as the sham group. The mean diameter of non-myelinated axonswas 0.96±0.33 μm. Comparing the VEGF-treated group to the castrated+LacZ group, the p values of myelinated axon, myelin sheath andnonmyelinated axon were 0.113, 0.05 and 0.000 respectively. The nervefibers and myelin sheath in the testosterone replacement group appearedsimilar to the sham group.

Intracavernosal Tissues

Intracavernous Smooth Muscle Cells:

In sham-operated rats, the smooth muscle cells (myocytes) were usuallyarranged in clusters and were separated by fine strands offibroconnective tissue. The cytoplasm of these myocytes containedabundant contractile myofilaments and dense bodies. Occasionally, smallaggregates of organelles, including mitochondria, rough endoplasmicreticulum and Golgi apparatus, were found adjacent to the nucleus. Thecell membrane (sarcolemma) consisted typically of alternating densebands and light bands. The light bands contain numerous pinocytoticvesicles (caveolae). The intercellular spaces among myocytes wereusually quite narrow with many gap junctions connecting individualcells. Nerve terminal varicosities were frequently seen located nearclusters of smooth muscle cells. In low power micrographs (6,500×) ofcastration with or without LacZ rats, the smooth muscle cells appearedscattered in a field of connective tissues. The major differencesbetween the castrated and castrated+testosterone-treated rats were theincrease in cytoplasmic myofilaments and the decrease in intercellularspaces in the latter group of rats. The myocytes in testosterone-treatedrats appeared packed in clusters rather than scattered.

Striking differences were noted when comparing the AAV-VEGF and VEGFprotein-treated rats to the castrated+Lac Z rats. The smooth muscleswere arranged in clusters with minimal intercellular spaces. Under highpower (9,400×), we noted the following: an increase in myofilaments anddense bodies, a decrease in dense bands, and an increase in the numberof caveolae within the light bands of the sarcolemma.

Endothelial Cells:

In sham-operated rats, the cavernous sinusoids were lined by intactendothelium, the cytoplasm of which contained numerous pinocytoticvesicles (caveolae), mitochondria, rough endoplasmic reticulum, andGolgi apparatus. The nuclei of the endothelial cells were occasionallyseen and appeared oval-shaped or elongated. In castration with orwithout LacZ rats, the appearance of the capillaries and cavernoussinusoidal endothelium was similar to the sham operated group. InAAV-VEGF and VEGF protein-treated rats, the nuclei of the endothelialcells lining most of the capillaries and sinusoids were plump and morenumerous, indicative of endothelial hypertrophy and hyperplasia.

Enzyme-Linked Immuno-Sorbent Assay:

The goal of using an adenovirus vector for delivering the VEGF gene isto transfect the penile tissue such that VEGF protein expression may beincrease in the penis. To document that the AAV-VEGF treated animals hadincreased VEGF expression in the penile blood, samples were taken fromthe penis (penile bleed following glans amputation) and compared with asample of systemic blood (from the abdominal aorta) for animals groupstreated with both AAV-VEGF (n=7) and AAV-LacZ (n=7) (Table 5). While themean VEGF titer in the systemic serum of animals that did not receivethe VEGF gene (AAV-LacZ group) is 9.5±12.6 pg/ml, the AAV-VEGF treatedanimals demonstrated a marked increase in VEGF titer at 23.3±9.6 pg/ml(p=0.04). Similarly, serum from the penile blood in the AAV-LacZ grouphad a VEGF titer of 13.6±11.4 pg/ml compared to a mean of 29.7±14.4pg/ml in the group receiving the intracavernosal VEGF gene (p=0.039).This difference is statistically significant suggesting that increasedVEGF expression is occurring in the penile tissue after treatment withAAV-VEGF. TABLE 5 Results of ELISA for VEGF protein (mean VEGF in pg/ml)in serum samples from animals treated with intracorporal AAV-VEGF (p =0.03) and AAV-LacZ (p = 0.07) AAV-VEGF-treated AAV-LacZ-treated animalsanimals Penile bleed 29.7(+/−14.4) 13.6(+/−11.4) Systemic blood23.3(+/−9.6)  9.5(+/−12.6)

Discussion

To test our hypothesis, we first developed an animal model of venogenicerectile ED (Experiment 1: Model validation). Mills et al^(10, 11, 26)have previously shown that rats develop venogenic erectile ED within 6-8days following castration. Animals receiving testosterone repletionmaintain an intact veno-occlusive mechanism after castration. Thesestudies were performed using ganglionic electro-stimulation to generatean erection and the penile response gauged with ICP monitoring duringeither cavemosometry¹¹ or a penile arterial inflow measurement using alaser Doppler flow.¹⁰ or goal was to devise a technique for evaluatingvenous leak in animals similar to the technique used in humans. For thisreason, erection was generated using pharmacologic agents (papavarine)instead of ganglionic electro-stimulation. The physiologic parameters(maintenance inflow rate and ICP drop rate) used to diagnose venous leakin humans^(27,28) were reproduced in a rat model. Using this technique,pharmacocavernosometric findings were determined in normal animals andanimals with venogenic and arteriogenic ED. This method was found to bea sensitive and reproducible technique to evaluate penile arterialinsufficiency and venous leak in a rat model.

This model was then used to evaluate the efficacy of VEGF, administeredintracorporally as recombinant protein or adeno-associated virus genevector, to prevent the development of venogenic erectile ED (Experiment2: Prevention trial). It has been previously shown that castrationinduces an involution of the prostate gland and its vasculature.¹³Furthermore, after testosterone replacement, endothelial cellproliferation is stimulated and both blood flow and vascular volumes arenormalized. After castration, prostatic VEGF synthesis is downregulated, as determined by RT-PCR, western blot and immunohistochemicalanalysis.¹² Also, testosterone induces VEGF synthesis, suggesting thatVEGF may be a tissue mediator of androgenic effects on the prostate. Thegoal of Experiment 2 was to determine if VEGF could prevent thedevelopment of venous leak in the rat model. Both testosteronereplacement and VEGF treatment maintained erectile function whenadministered immediately after castration. Animal groups receiving notestosterone replacement or intracorporal AAV-LacZ showed persistentvenogenic erectile ED after castration. Histological examination ofsmooth muscle content and morphology revealed deterioration in both thequality and quantity of penile smooth muscle after castration. Electronmicroscopic examination also revealed alteration of cell membrane andwidening of intercellular spaces. Smooth muscle content as measured byalpha actin staining was normalized in animals receiving eithertestosterone or VEGF, evidence of preserved smooth muscle integrity withsuch preventative treatment.

The final phase was a treatment trial (Experiment 3) in which animalswere first documented to have venous leak, 6 weeks after castration, andthen treated with intracorporal VEGF protein. One month after suchtreatment, cavernosometry was repeated and restoration of near normalerectile function was found. We believe that this is the firstexperimental evidence of any medical therapy improving venogenicerectile ED. The exact mechanism by which VEGF improves erectilefunction is unknown. Nevertheless, we observed clear evidence ofrestoration of neural and smooth muscle integrity as well as hyperplasiaand hypertrophy of endothelial cells after VEGF treatment. Conceivably,increased cavernosal neovascularity may lead to functional or structuralchanges in the nerve and smooth muscles. Alternatively, the directeffect of VEGF on the nerve and smooth muscle may also play a role sinceVEGF has been reported to have a direct trophic effect on the penilesmooth muscle cells and spinal neurons in culture 14-16.

Nerve function and NOS expression is depressed with androgen ablation,and this may be another target for VEGF action in the penis. Furtherstudies are underway to study the mechanism of VEGF action in the penis.

Conclusion

The technique presented here for pharmacologic cavernosometry is asimple and reproducible method to evaluate vasculogenic ED in a ratmodel. Normal erectile function and ED due to arterial insufficiency orvenous leak may be diagnosed by characteristic cavernosometric findings.Using this technique, the presence of veno-occlusive disease may bediagnosed in animals 6 weeks after either castration or ligation of theinternal iliac arteries.

Animals treated with testosterone replacement at the time of castrationretain normal erectile function while those without testosteronereplacement develop venous leak. If these animals are treated withintracavernosal recombinant VEGF protein or AAV-VEGF at the time ofcastration, their erectile function is maintained and venous leak isprevented. The mechanism for this is not known at present, but we find adecrease in penile smooth muscle content in the castrated group comparedwith either the testosterone replacement group or the group receivingintracavernosal VEGF or AAV-VEGF. Penile smooth muscle morphology isuniformly degenerated after castration. Animals treated with theintracorporal AAV-VEGF transfection vector demonstrate significantlymore VEGF protein in their penile serum compared to the systemic serum,and markedly more that control animals, indicating an increasedexpression of penile VEGF in these animals.

When rats with established venogenic ED are treated with one dose ofintracavemosal recombinant VEGF protein, their erectile function returnsto nearly normal, with reversal of the veno-occlusive defect.Electronmicroscopy revealed endothelial cell hyperplasia and hypertrophyas well as restoration of smooth muscle and neural integrity in thepenile tissue after VEGF treatment. Since impairment of erectile nerve,endothelial cell, and the cavernous smooth musculature is the finalcommon pathway of various type of organic ED, VEGF therapy may hold thekey to prevention and cure of many forms of ED.

References

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L., O'Hanlon, J. K. & Sachs, B. D.: Differential    maintenance of penile responses and copulatory behavior by gonadal    hormones in castrated male rats. Horm Behav, 18:, 56, 1984.-   7. Zvara, P., Sioufi, R., Schipper, H. M. et al.: Nitric oxide    mediated erectile activity is a testosterone dependent event: a rat    erection model. Int J Impot Res, 7:, 209, 1995.-   8. Lekas, E., Johansson, M., Widmark, A. et al.: Decrement of blood    flow precedes the involution of the ventral prostate in the rat    after castration. Urol Res, 25:, 309, 1997.-   9. Haggstrom, S., Wikstrom, P., Bergh, A. et al.: Expression of    vascular endothelial growth factor and its receptors in the rat    ventral prostate and Dunning R3327 PAP adenocarcinoma before and    after castration. Prostate, 36:, 71, 1998.-   10. Mills, T. M., Lewis, R. W. & Stopper, V. S.: Androgenic    maintenance of inflow and veno-occlusion during erection in the rat.    Biol Reprod, 59:, 1413, 1998.-   11. Mills, T. M., Stopper, V. S. & Wiedmeier, V. T.: Effects of    castration and androgen replacement on the hemodynamics of penile    erection in the rat. Biol Reprod, 51:, 234, 1994.-   12. Haggstrom, S., Lissbrant, I. F., Bergh, A. et al.: Testosterone    induces vascular endothelial growth factor synthesis in the ventral    prostate in castrated rats. J Urol, 161:, 1620, 1999.-   13. Franck-Lissbrant, I., Haggstrom, S., Damber, J. E. et al.:    Testosterone stimulates angiogenesis and vascular regrowth in the    ventral prostate in castrated adult rats [see comments].    Endocrinology, 139:, 451, 1998.-   14. Liu X, Lin C-S, Graziottin T, Resplande J and Lue T F: Vascular    endothelial growth factor promotes proliferation and migration of    cavernous smooth muscle cells. J Urol; 166: 354-360, 2001.-   15. Jin K L, Mao X O, Greenberg D A.: Vascular endothelial growth    factor: direct neuroprotective effect in vitro ischemia. Proc Natl    Acad Sci USA. 97:10242-7, 2000.-   16. Sandal M, Sunder F, Kanji M. Vascular endothelial growth factor    is a neurotrophic factor which stimulates axonal outgrowth through    the flk-1 receptor. Eur J Neurosci: 2000 12:4243-54, 2000.-   17. Verdonck, A., De Ridder, L., Kuhn, R. et al.: Effect of    testosterone replacement after neonatal castration on craniofacial    growth in rats. Arch Oral Biol, 43:, 551, 1998.-   18. Parker, C. R., Jr., Ellegood, J. O. & Mahesh, V. B.: Methods for    multiple steroid radioimmunoassay. J Steroid Biochem, 6:, 1, 1975.-   19. Maeda, Y., Ikeda, U., Ogasawara, Y. et al.: Gene transfer into    vascular cells using adeno-associated virus (AAV) vectors.    Cardiovasc Res, 35:, 514, 1997.-   20. Dong, J. Y., Fan, P. D. & Frizzell, R. A.: Quantitative analysis    of the packaging capacity of recombinant adeno-associated virus. Hum    Gene Ther, 7:, 2101, 1996.-   21. Ferrara, N., Houck, K., Jakeman, L. et al.: Molecular and    biological properties of the vascular endothelial growth factor    family of proteins. Endocr Rev, 13:, 18, 1992.-   22. Stenberg, P. E., Shuman, M. A., Levine, S. P. et al.:    Redistribution of alpha-granules and their contents in    thrombin-stimulated platelets. J Cell Biol, 98:, 748, 1984.-   23. Karadeniz, T., Topsakal, M., Aydogmus, A. et al.: Correlation of    ultrastructural alterations in cavernous tissue with the clinical    diagnosis vasculogenic impotence. Urol Int, 57:, 58, 1996.-   24. Baumgartner, I., Pieczek, A., Manor, O. et al.: Constitutive    expression of phVEGF165 after intramuscular gene transfer promotes    collateral vessel development in patients with critical limb    ischemia [see comments]. Circulation, 97:, 1114, 1998.-   25. Symes, J. F., Losordo, D. W., Vale, P. R. et al.: Gene therapy    with vascular endothelial growth factor for inoperable coronary    artery disease. 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EXAMPLE 3 The Effect of Adeno-Associated Virus-Mediated Brain-DerivedNeurotrophic Factor (BDNF) in an Animal Model for Neurogenic Impotence

Animals

Male Sprague-Dawley rats (N=34; age, 3 months; weight, 350 to 400 gm.)were divided into two groups: sham (N=10) and experimental (N=24). Therats in the sham group underwent periprostatic dissection andidentification of bilateral cavernous nerves without other manipulation;those in the experimental groups underwent bilateral cavernous nervefreezing. Several minutes after surgery, half of the experimentalanimals (LacZ group, N=12) received intracavernous AAV-LacZ injection,and the remainder (BDNF group, N=12) received AAV-BDNF. Half of the ratsin each group were sacrificed at week 4 and the rest at week 8 forcollection of penile tissue. In all animals, erectile function wasassessed by electrostimulation of the cavernous nerves before sacrifice.

Surgical Procedure and Transfection Technique

Under intraperitoneal pentobarbital sodium anesthesia (50 mg./kg.), eachanimal was placed on a heating pad to maintain its body temperature at37° C. Through a lower abdominal midline incision, the areaposterolateral to the prostate was explored. The major pelvic gangliaand the cavernous nerve were identified with an operating microscope(Olympus, 10-40×). In the experimental group, the cavernous nerve wasfrozen bilaterally for 1 min. with a thermocouple used to control thetemperature (5 mm. diameter, Omega HH21 handheld microprocessor digitalthermometer). (Before surgery, the thermocouple had been placed in a15-ml. disposable centrifuge tube filled with ground dry ice and kept ina thermo-flask [Lab-Line Instruments Inc.] filled with dry ice.) Thetemperature of the probe at the beginning of the procedure was −80° C.,increasing to −50° C. at 1 min. To prevent disruption of the nerve, 0.2ml. saline was used to disengage the tip of the probe from the nervebefore removal.

After the freezing procedure, the right side of the proximal crus wasexposed, and 0.05 ml. of either 10¹⁰ AAV-LacZ or 10¹⁰ AAV-BDNF wasinjected into 12 rats each through a tuberculin syringe with a 30Gneedle.

Preparation of AAV-BDNF

Cloning of BDNF cDNA:

We used RT-PCR to identify BDNF expression in a human neuroblastoma cellline, SK-N-BE(2). We then used a primer pair,5′-CCCTACAGGTCGACCAGGTGA-3′ (SEQ ID NO:1) and5′-CTATACAACATGGATCCACTA-3′ (SEQ ID NO:2), to amplify the codingsequence of BDNF from SK-N-BE(2) cDNA (underlined sequences are designedXhoI and BamHI restriction sites, respectively). After digestion withShoI and BamHI, the amplified product was cloned into pBluescript(Stratagene Inc., La Jolla, Calif.) and fully sequenced. The BDNF cDNAwas then re-cloned into pcDNA4, a modified version of pcDNA3 plasmid(Invitrogen, Inc., Carlsbad, Calif.) that contains the cytomegalovirus(CMV) promoter for driving the expression of BDNF in mammalian cells.

Construction of rAAV-BDNF:

The above pcDNA4BDNF was digested with Sal1 to release the expressioncassette containing cytomegalovirus immediate-early (CMVie) promoter,BDNF gene and bovine growth hormone (BGH) poly-A signal. This expressioncassette was inserted into an AAV vector, pAV53, resulting in theconstruction of rAAV-BDNF.

Virus Production and Titration:

rAAV-BDNF was produced by a three-plasmid co-transfection method. Twenty15-cm plates of 293 cells (50 to 60% confluent) were maintained inDulbecco modified Eagle medium (DMEM, Gibco) supplemented with 10% fetalbovine serum (Hyclone) and 25 mM. HEPES and co-transfected by thecalcium phosphate method with a total of 45 μg. DNA, 15 μg. of AAV-BDNFvector, and 15 μg. each of pLHP19 (AAV helper plasmid) and pLadeno5(adenovirus helper plasmid), kindly provided by AVIGEN. Six hours aftertransfection, the medium was replaced with fresh DMEM containing 1%fetal bovine serum. The cells were harvested at 48 h posttransfection bycentrifugation (1000 g for 10 min.), and the cell pellets werere-suspended in 0.1 M. Tris-HCL, 0.15 M. NaCl solution (pH 8.0) andsubjected to four cycles of freeze-thaw and removal of cell debris.Large-scale rAAV CsCl purification was carried out as describedpreviously.¹² AAV-BDNF vector titer was determined by quantitative dotblot hybridization of DNase-treated stocks. The AAV vector titer used inthe experiment refers to the particle number of AAV vector genomes inthe sample as determined by the quantitative dot blot assay.

Functional Evaluation and Tissue Procurement

At weeks 4 and 8 postoperatively, rats in each group were re-exploredfor direct electrostimulation of the cavernous nerves before tissuecollection. The skin overlying the penis was incised and theischiocavernous muscle was partly removed to expose both penile crura. A23G butterfly needle connected to PE-50 tubing was inserted in the rightcrus for pressure measurement. Electrostimulation was performed with adelicate stainless-steel bipolar hook electrode attached to amulti-jointed clamp. (Each pole was 0.2 mm. in diameter; the two poleswere separated by 1 mm.) Short wave pulses were generated by a Macintoshcomputer with a custom-built constant current amplifier. Stimulusparameters were 1.5 mA., frequency 20 Hz., pulse width 0.2 m sec.,duration 50 sec. Each cavernous nerve was stimulated and intracavernouspressures were measured and recorded with a Macintosh computerprogrammed with LabVIEW 4.0 software (National Instruments, Austin,Tex.). The pressure for each animal was determined by the mean of bothsides.

NADPH Diaphorase Staining

After sacrifice, samples of major pelvic ganglia and penile tissue werefixed for 3 hours in a cold, freshly prepared, solution of 2%formaldehyde, 0.002% picric acid in 0.1 M. phosphate buffer, pH 8.0.Tissues were cryoprotected for 24 hours in cold 30% sucrose in 0.1 M.phosphate buffer, pH 8.0. They were then embedded in O.C.T. compound(Tissue-Tek, Miles Laboratory), frozen in liquid nitrogen, and stored at−70° C. Cryostat tissue sections were cut at 10 μm., adhered to chargedslides, air-dried for 5 min., and hydrated for 5 min. with 0.1 M. PO₄,pH 8.0. Sections were incubated with 0.1 mM. NADPH, 0.2 mM. nitrobluetetrazolium, 0.2% Triton X-100 in 0.1 M. PO₄, pH 8.0, for 60 min. atroom temperature. The reaction was terminated by washing in buffer.Slides were then coverslipped with buffered glycerin as the mountingmedium.¹³

The presence of NADPH diaphorase-positive nerves was evidenced as a bluestain in the major pelvic ganglia, dorsal nerves and cavernous tissue,and the staining pattern was assessed by counting the number of positiveneurons in 4 random fields (magnification 400×). The percentage ofdarkly and lightly stained cells in the major pelvic ganglia wascalculated by dividing the number of these cells by the total number ofpositive cells.

Nitric Oxide Synthase Antibody Staining

Tissue fixation was the same as with NADPH-diaphorase specimens. Afterfreezing, 10-μm. cryostat tissue sections were adhered to chargedslides, air-dried, and hydrated for 5 min. with 0.05M. sodium phosphatebuffer (PBS, pH 7.4). Sections were treated with hydrogenperoxide/methanol to quench endogenous peroxidase activity. Afterrinsing with water, sections were washed twice in PBS for 5 min.followed by 30 min. of room-temperature incubation with 3% horseserum/PBS/0.3% triton X-100. After draining solution from sections,tissues were incubated for 60 min. at room temperature with mousemonoclonal anti-nNOS (Transduction Laboratories, Lexington, Ky.) at a1:500 dilution. After washing for 5 min. with PBS/TX and then for 5 min.twice with PBS alone, sections were immunostained with theavidin-biotin-peroxidase method (Elite ABC, Vector Labs, BurlingameCalif.), with diaminobenzidine as the chromagen, followed bycounterstaining with hematoxylin.

Statistical Analysis: The nonparametric Mann-Whitney U and ANOVA testswith Statview 4.02 software were used to compare results. Values wereconsidered significant at p<0.05.

Results

Functional Studies

In both the LacZ and BDNF groups, maximal intracavernous pressure inresponse to bilateral cavernous nerve electrostimulation was less thanin the sham group. However, the pressure in the BDNF group wassignificantly higher than in the LacZ group at both 4 and 8 weeks (FIG.1 and Table 6). TABLE 6 Maximal intracavernous Pressure In Response toElectrostimulation 4 and 8 Weeks After Bilateral Cavernous NerveFreezing Time Sham Operation LacZ BDNF (Weeks) (n = 10) (n = 12) (n =12) 4   105 ± 10.5* 28.4 ± 5.5 585 ± 11.7† 8 115.5 ± 7.7 37.7 ± 7.9 61.3± 12.5†*All values are expressed as cm H2O; mean ± SD†p < 0.05 v. LacZ.NADPH Diaphorase Staining

Dorsal Nerve:

At week 4, the LacZ group showed significantly fewer NADPHdiaphorase-positive nerve fibers than did the BDNF group. At week 8, thenumber had increased in both groups, but there were still significantlyfewer in the LacZ group When compared with the sham group, bothexperimental groups showed fewer positive nerve fibers (Table 7). TABLE7 NADPH Diaphorase-positive Nerve Fibers In the Dorsal Nerve andCavernous Tissue Time Sham Operation LacZ BDNF (Weeks) (N = 10) (N = 12)(N = 12) Dorsal Nerve 4 Weeks 138.5 ± 10.5* 45.7 ± 5.8 65.5 ± 15.5^(†) 8Weeks 140.2 ± 9.8 55.6 ± 8.4 86.4 ± 12.2^(†) Cavernous Tissue 4 Weeks  103 ± 9.8 25.5 ± 3.6 45.5 ± 10.5^(†) 8 Weeks 110.2 ± 10.5 35.6 ± 10.466.1 ± 15.2^(†)*All values expressed as Mean ± S.D.^(†)p < 0.05 vs. LacZ

Intracavernous Nerves:

Histologic evaluation showed significantly fewer NADPHdiaphorase-positive nerve fibers in the trabecular smooth muscle of theLacZ group than in the BDNF group at both 4 and 8 weeks. When comparedwith the sham group, both experimental groups had significantly fewer(Table 7).

Major Pelvic Ganglia:

At week 4, most of the neurons in the major pelvic ganglia in the LacZgroup exhibited a lighter staining pattern than that seen in the BDNFgroup. In addition, at 8 weeks most of these LacZ neurons had irregularcell contour and multiple vacuoles in the cytoplasm. The percentage ofdarkly stained cells in the BDNF group at both time points wassignificantly higher than in the LacZ group, and their appearance (i.e.smooth contour and very few vacuoles in the cytoplasm) was similar tothat in the sham group. In both experimental groups, the percentage ofdarkly stained cells was significantly less than in the sham group(Table 8). TABLE 8 NADPH Diaphorase-positive Neurons in the Major PelvicGanglion % % Time Group Dark Stain Light Stain Total Dark Stain LightStain 4 Weeks Sham  102 ± 0.5* 21.7 ± 3.5 123.5 ± 4.5† 82.2 ± 5.3 17.3 ±6.8 LacZ 32.4 ± 15.2 52.5 ± 20.4   85 ± 20.1 38.5 ± 17.2 60.7 ± 17.2BDNF 61.8 ± 23.7 33.8 ± 9  95.7 ± 22.4 62.1 ± 13.1‡ 38.6 ± 11.8 8 WeeksSham  101 ± 12.3 25.5 ± 5.5 136.7 ± 11.3† 80.7 ± 9.8 19.2 ± 4.5 LacZ  45 ± 12.7 53.3 ± 9.5  98.5 ± 14.3 45.8 ± 0.8 54.1 ± 0.8 BDNF 69.4 ±21.1 38.4 ± 14 107.8 ± 23.58 64.3 ± 10.7‡ 35.6 ± 10.7*All values expressed as Mean ± S.D.†p < 0.05 vs. either experimental group‡p < 0.05 vs. LacZ groupnNOS Immunostaining

Immunostaining of penile tissue and the major pelvic ganglia for nNOSrevealed positive staining in the same nerve fibers and neurons as withNADPH diaphorase. The neurons of the major pelvic ganglia of the LacZgroup showed lighter staining patterns and many more vacuoles in thecytoplasm than did the neurons in the BDNF and sham groups.

Discussion

The aim of the present study was to investigate the feasibility of usingAAV-BDNF gene transfer to facilitate recovery of potency after bilateralcavernous nerve injury. Our past studies have led us to believe thatbilateral cavernous nerve freezing in the rat is a suitable model forsuch injury because the neural sheath is preserved, as it is in patientsundergoing nerve-sparing prostatectomy or cryoablation. In addition, thecourse and extent of functional recovery have been well documented inthis model.¹⁴ We used maximal intracavernous pressure in response tocavernous nerve electrostimulation to assess recovery of erectilefunction. Although apomorphine-induced erection may be more physiologic,we do not believe it can reliably differentiate partial from fullerection in rats with cavernous nerve injury.

In this study, the number of nNOS-containing neurons in the major pelvicganglia of both experimental groups was less than in the sham group.However, the percentage of darkly stained neurons was significantlygreater in the BDNF group than in the LacZ group at both 4 and 8 weeks.Moreover, most neurons of the BDNF and sham groups did not show thecytoplasmic vacuoles and irregular cell contour seen in the LacZ group.These findings suggest that the production of BDNF protein in peniletissue can be retrogradely transported to the major pelvic ganglia toprevent neuronal damage and preserve nNOS enzymes in the neurons. Thisin turn facilitates the recovery of erectile function, as evidenced bythe more numerous nNOS-positive nerve fibers in the erectile tissue andhigher intracavernous pressure in the BDNF group.

A previous study has shown a significantly increased survival ofmotoneurons 1 week after axotomy in animals pretreated withadenovirus-encoding BDNF or glial cell line-derived neurotrophic factor(GDNF).¹⁷ However, because of the disadvantages of the adenovirus, weused adeno-associated virus (AAV), a unique member of the non-enveloped,single-stranded-DNA Parvovirus that possesses several properties thatdistinguish it from other gene-transfer vectors. Its advantages includestable and efficient integration of viral DNA into the host genome,¹⁸lack of associated human disease,¹⁹ broad host range, ability to infectgrowth-arrested cells,²⁰ and ability to carry non-viral regulatorysequences without interference from the viral genome.²¹ In addition, nosuperinfection inhibition is associated with AAV vectors.¹⁸ The infectedcells are spread several millimeters around the needle tract. AAV isable to infect axon terminals and is retrogradely transported. Injectionof AAV vector expressing LacZ into several brain regions has shown thepresence of transgene expression as early as 24 hours,²² lasting (atsignificantly decreased levels) as long as 6 months.

The penis is a convenient organ for gene therapy because of its externallocation and slow circulation in the flaccid state. In addition, itssinusoidal structure and the gap junctions between smooth muscles ensurewide distribution of injected vectors. To our knowledge, this is thefirst demonstration of gene therapy with AAV-BDNF used to facilitate therecovery of nNOS-containing nerves and neurons and consequent erectilefunction. Because our previous studies have shown that 3 to 6 months maybe required for more complete regeneration of cavernous nerves anderectile function in both unilateral resection and unilateral freezingmodels,^(3,16) we are presently conducting a further study to examinethe effects of higher AAV-BDNF titer and longer follow-up in this model.

Conclusion

Our results showed that intracavernous injection of AAV-BDNF afterfreezing of bilateral cavernous nerves had the following effects: 1)facilitated the recovery of erectile function, 2) enhanced theregeneration of the intracavernous and dorsal nerves, and 3) preventedneuronal degeneration in the major pelvic ganglia. If further studiesconfirm its effectiveness and safety, intracavernous injection ofneurotrophins or other growth factors has the potential to be a curativetherapy for neurogenic erectile dysfunction after cryoablation orradical pelvic surgery. Bakircioglu ME, et al., J Urol. 165:2103-9(2001).

References

-   1. Walsh, P. C., and Mostwin, J. L. Radical prostatectomy and    cystoprostatectomy with preservation of potency. Results using a new    nerve-sparing technique. Br. J. Urol., 56: 694, 1984.-   2. Paick, J. S., Donatucci, C. F., and Lue, T. F. Anatomy of    cavernous nerves distal to prostate: microdissection study in adult    male cadavers. Urology, 42: 145, 1993.-   3. Carrier, S., Zvara, P., Nunes, L., Kour, N. W., Rehman, J., and    Lue, T. F. Regeneration of nitric oxide synthase-containing nerves    after cavernous nerve neurotomy in the rat. J. Urol. 153: 1722,    1995.-   4. Jung, G. W., Spencer, E. M., and Lue, T. F. Growth hormone    enhances regeneration of nitric oxide synthase-containing penile    nerves after cavernous nerve neurotomy in rats. J. Urol. 160: 1899,    1998.-   5. Leibrock, J., Lottspeich, F., Hohn, A., Hofer, M., Hengerer, B.,    Masiakowski, P., Thoenen, H., and Barde, Y. A. Molecular cloning and    expression of brain-derived neurotrophic factor. Nature. 341: 149,    1989.-   6. Ide, C.: Peripheral nerve regeneration. Neurosci. Res. 25: 101,    1996.-   7. DiStefano, P. S., Friedman, B., Radziejewski, C., Alexander, C.,    Boland, P., Schick, C. M., Lindsay, R. M., and Wiegand, S. J. The    neurotrophins BDNF, NT-3, and NGF display distinct patterns of    retrograde axonal transport in peripheral and central neurons.    Neuron. 8: 983, 1992.-   8. Oppenheim, R. W., Yin, Q. W., Prevette, D., and Yan, Q.    Brain-derived neurotrophic factor rescues developing avian    motoneurons from cell death. Nature. 360: 755, 1992.-   9. Yan, Q., Elliott, J., and Snider, W. D. Brain-derived    neurotrophic factor rescues spinal motor neurons from    axotomy-induced cell death. Nature. 360: 753, 1992.-   10. Meyer, M., Matsuoka, I., Wetmore, C., Olson, L., and Thoenen, H.    Enhanced synthesis of brain-derived neurotrophic factor in the    lesioned peripheral nerve: different mechanisms are responsible for    the regulation of BDNF and NGF mRNA. J. Cell Biol. 119: 45, 1992.-   11. Nonomura, T., Nishio, C., Lindsay, R. M., and Hatanaka, H.    Cultured basal forebrain cholinergic neurons from postnatal rats    show both overlapping and non-overlapping responses to the    neurotrophins. Brain Res. 683: 129, 1995.-   12. Matsushita, T., Elliger, S., Elliger, C., Podsakoff, G.,    Villarreal, L., Kurtzman, G. J., Iwaki, Y., and Colosi, P.    Adeno-associated virus vectors can be efficiently produced without    helper virus. Gene Ther. 5: 938, 1998.-   13. Alm, P., Larsson, B., Ekblad, E., Sundler, F., and    Andersson, K. E. Immunohistochemical localization of peripheral    nitric oxide synthase-containing nerves using antibodies raised    against synthesized C- and N-terminal fragments of a cloned enzyme    from rat brain. Acta Physiol. Scand. 148: 421, 1993.-   14. Korsching, S. The neurotrophic factor concept: a    reexamination. J. Neurosci. 13: 2739, 1993.-   15. Lewin, G. R. and Barde, Y. A. Physiology of the neurotrophins.    Annu. Rev. Neurosci. 19: 289, 1996.-   16. El-Sakka, A. I., Hassan, M. U., Selph, C., Perinchery, G.,    Dahiya, R., and Lue, T. F. Effect of cavernous nerve freezing on    protein and gene expression of nitric oxide synthase in the rat    penis and pelvic ganglia. J. Urol. 160: 2245, 1998.-   17. Gimenez y Ribotta, M., Revah, F., Pradier, L., Loquet, I.,    Mallet, J., and Privat, A. Prevention of motoneuron death by    adenovirus-mediated neurotrophic factors. J. Neurosci. Res. 48: 281,    1997.-   18. McLaughlin, S. K., Collis, P., Hermonat, P. L., and Muzyczka, N.    Adeno-associated virus general transduction vectors: analysis of    proviral structures. J. Virol. 62: 1963, 1988.-   19. Bems, K. I., Cheung, A., Ostrove, J., and Lewis, M.:    Adeno-associated virus latent infection. In: B. W. J. Mahy, A. C.    Minson, and G. K. Darby (eds.), Virus Persistence. Cambridge, UK:    Cambridge University Press, 1982.-   20. Podsakoff, G., Wong, K. K., Jr., and Chatterjee, S. Efficient    gene transfer into nondividing cells by adeno-associated virus-based    vectors. J. Virol. 68: 5656, 1994.-   21. Miller, J. L., Walsh, C. E., Ney, P. A., Samulski, R. J., and    Nienhuis, A. W. Single-copy transduction and expression of human    gamma-globin in K562 erythroleukemia cells using recombinant    adeno-associated virus vectors: the effect of mutations in NF-E2 and    GATA-1 binding motifs within the hypersensitivity site 2 enhancer    [published erratum appears in Blood 1995 Feb. 1; 85(3):862]. Blood.    82: 1900, 1993.-   22. During, M. J. and Leone, P. Adeno-associated virus vectors for    gene therapy of neurodegenerative disorders. Clin. Neurosci. 3: 292,    1995.

EXAMPLE 4 Vascular Endothelial Growth Factor Promotes Proliferation andMigration of Cavernous Smooth Muscle Cells

Animals

Male Wistar rats were obtained from Charles River Laboratories(Wilmington, Mass.). Young rats were 1, 2, 3, 4, 6, 11, and 16 weeks ofage. Old rats were 28 months of age.

Regents

All chemicals were from Sigma-Aldrich Co. (St. Louis, Mo.) unless notedotherwise. Recombinant human VEGF₁₆₅ was from Calbiochem BiosciencesInc. (La Jolla, Calif.). Fetal bovine serum (FBS) and Trypsin-EDTA werefrom Life Technologies, Inc. (Grand Island, N.Y.). All other cellculture regents were obtained from Cell Culture Facility, University ofCalifornia, San Francisco.

Cell Culture

Each primary culture of CSMC was prepared from the corpora cavernosa of2-3 rats by the following procedure. The penis was cleared of theurethra, blood vessels, fat and connective tissue. The remaining smoothmuscle tissue was washed 3 times in sterile PBS (phosphate-bufferedsaline) and cut into 2-3 mm³ segments. The segments were placed evenlyonto a 100-mm cell culture dish (Falcon-Becton Dickinson Labware,Franklin Lakes, N.J.) inside a cell culture hood. Approximately 10 minlater, 10 ml of Dulbecco's Modified Eagle Medium (DMEM) containingpenicillin (100 units/ml), streptomycin (100 μg/ml), and 10% FBS wascarefully pipetted into the dish. The dish was then kept undisturbed ina humidified 37° C. incubator with 5% CO₂. Five days later, tissuesegments that have detached from the dish were removed, and the mediumwas replaced with fresh medium. Another 5 days later, all tissuesegments were removed and the medium was again replaced with freshmedium. When small islands of cells were noticeable, they weretrypsinized and transferred to a fresh culture dish. Expansion of eachcell strain was continued with change of medium every 3 days andpassages (trypsinization and seeding) approximately every 10 days. Allcells used in the following experiments were from passages 4 through 10.Primary aorta SMC cultures were prepared similarly with aortas isolatedfrom 16-week-old male rats. All cell cultures were confirmed for theirsmooth muscle identity by an indirect immunofluorescence staining withan anti-smooth muscle myosin heavy chain antibody (Sigma-Aldrich Co. St.Louis, Mo.).

Quantification of VEGF

Each CSMC from rats of different ages was seeded at 4×10⁵ cells per wellin 3 ml of DMEM with 10% FBS in 6-well culture plates. Seventy-two hrlater, the medium was removed for quantification of VEGF and the cellswere trypsinized for the determination of cell number. For quantifyingVEGF in the medium, the Mouse VEGF Immunoassay Kit (R&D Systems,Minneapolis, Minn.), which reacts with rat VEGF but not with bovineVEGF, was used. All assays were performed in duplicate in eachexperiment and all data presented in the Results section are the averageof three independent experiments.

Proliferation Assay

Cell proliferation assays were performed with the CellTiter-96 kit fromPromega Inc. (Madison, Wis.). Each SMC strain from different-aged ratswas assayed in one flat-bottom 96-well cell culture plate. The plate wasdivided into 12 rows that contained VEGF at concentrations from 0 to 100ng per ml of serum-free DMEM (supplemented with 0.1% BSA). Each well inthe same row received 50 μl of the medium containing the sameconcentration of VEGF. Thereafter, SMC that were grown to 70% confluencewere rinsed twice with PBS, trypsinized, and resuspended in serum-freeDMEM (supplemented with 0.1% BSA) at 100,000 cell per ml. Aliquots of 50μl of the cell suspension were then transferred to the 96-well plate sothat each well contained 5,000 cells in a final volume of 100 μl. Theplate was incubated in a 37° C., humidified incubator with 5% CO₂. Threedays later, 20 μl of CellTiter 96®AQueous One Solution Reagent was addedto each well. After 4 hr of further incubation at 37° C. in thehumidified, 5% CO₂ incubator, color development, which reflects cellnumbers, was recorded with a plate reader (Molecular Devices Corp.,Sunnyvale, Calif.) at 490-nm absorbance. For proliferation assaysconcerning concentrations of FBS in the growth medium, the assayprocedure was the same except that different amounts of FBS, instead ofVEGF, were added to the medium. All assays were performed in duplicatein each experiment and all data presented in the Results section are theaverage of three independent experiments.

Migration Assay

Cell migration assays were performed in 6.5-mm Transwell chambers ofCorning Costar Corporation (Cambridge, Mass.). The Transwell inserts(upper chambers) were bathed in a solution containing 13.4 μg/mlfibronectin in PBS at 37° C. for 1 hr and allowed to air-dry. The driedupper chambers were then placed in the lower chambers, each of whichcontained 700 μl of serum-free DMEM supplemented with 0.1% BSA and 0 to500 ng/ml of VEGF. SMC that had been grown to 70% confluence werefurther conditioned in serum-free DMEM (containing 0.1% BSA) for 2 hrand then trypsinized. The trypsinized cells were washed in PBS andresuspended at a concentration of 80,000 cells per ml in serum-free DMEM(containing 0.1% BSA). One hundred μl of the cell suspension was thenadded to each upper chamber. After 4 hr of incubation at 37° C., allliquid in the upper and lower chambers was removed by aspiration. Themembranes in the upper chambers were subsequently fixed in 1% bufferedformalin for 5 min and stained with 2% crystal violet. Non-migratorycells on the upper side of the membranes were scraped off with cottonswabs. Well locations were marked on the membranes and total cells perwell were counted visually in a masked fashion. Well locations were thencorrelated with the concentrations of VEGF in the wells. All assays wereperformed in duplicate in each experiment and all data presented in theResults section are the average of three independent experiments.

RNA Preparation

Cultured cells and rat tissues were homogenized in Tri-Reagent RNAextraction solution (Molecular Research Center, Cincinnati, Ohio).Following the recommended procedure by the supplier, RNAs were furthertreated with DNase I to remove traces of contaminating DNA. Quantity andintegrity of RNAs were examined by spectrophotometry and agarose gelelectrophoresis, respectively. Human heart RNAs were purchased fromClontech Laboratories, Inc. (Palo Alto, Calif.).

RT-PCR Analysis

RT-PCR (reverse transcription-polymerase chain reaction) was performedin an RT step and a PCR step. In the RT step, the cellular mRNAs werereverse-transcribed into a “library” of complementary DNAs (cDNAs). ThiscDNA library was then used for the analysis of various genes in the PCRstep. The RT procedure was performed with the SuperScript reversetranscriptase (Life Technologies, Inc., Gaithersburg, Md.) and itsaccompanying reagents. Briefly, 2.5 μg of each tissue RNA was annealedto 0.4 μg of oligo-dT primer in a 12 μl volume. Four μl of 5× buffer, 2μl of 0.1 M DTT, 1 μl of 10 mM dNTP, and 1 μl of SuperScript reversetranscriptase were then added to bring the final reaction volume to 20μl. After one hour of incubation at 42° C., the RT mixture was incubatedat 70° C. for 10 min to inactivate the reverse transcriptase. Eighty μlof TE buffer was then added to make a 5× diluted library. A portion ofthis library was further diluted to various concentrations (up to 100×dilution). One μl of each dilution was then used in a 10 μl PCR toidentify the optimal input within the linear amplification range. Inaddition to the 1 μl diluted library, the PCR mixture consisted of 10 ngof each of a primer pair and reagents supplied with the Taq polymerase(Life Technologies, Inc., Gaithersburg, Md.). PCR was performed in theDNA Engine thermocycler (MJ Research, Inc., Watertown, Mass.) undercalculated temperature control. The cycling program was set for 35cycles of 94° C., 5 sec; 55° C., 5 sec; 72° C., 10 sec, followed by onecycle of 72° C., 5 min. The PCR products were electrophoresed in 1.5%agarose gels in the presence of ethidium bromide, visualized by UVfluorescence, and recorded by a digital camera connected to a computer.TABLE 9 Oligonucleotide primers Size of Primer PCR Gene name Sequenceproduct β-Actin Actin-s 5′-TCTACAATGAGCTGCGTGTG-3′ 368 bp (SEQ ID NO:3)Actin-a 3′-AATGTCACGCACGATTTCCC-5′ (SEQ ID NO:4) VEGFR-1 VEGFR-1s5′-ATGCTGGATTGCTGGCACA-3′ 323 bp (SEQ ID NO:5) VEGFR-1A3′-TCAAACATGGAGGTGGCATT-5′ (SEQ ID NO:6) VEGFR-2 VEGFR-2s5′-GCCTTTGGCCAAGTGATTGA-3′ 479 bp (SEQ ID NO:7) VEGFR-2a3′-TCCAAGGTCAGGAAGTCCTT-5′ (SEQ ID NO:8)Oligonucleotide Primers

Primer pairs for RT-PCR analysis of VEGFR-1, VEGFR-2 and β-actin genesare listed in Table 9. They were designed to recognize the respectivemRNAs in both humans and rats.

Western Blot Analysis

Cultured cells were lysed in a buffer containing 1% IGEPAL CA-630, 0.5%sodium deoxycholate, 0.1% SDS, 10 μg/ml aprotinin, 10 μg/ml leupeptin,and 1×PBS. Cell lysates containing indicated amounts of protein wereelectrophoresed in 7.5% SDS-PAGE and then transferred to PVDF membrane.The membrane was stained with Ponceau S to verify the integrity of thetransferred proteins and to monitor the unbiased transfer of all proteinsamples. Detection of VEGFR-1 protein on the membrane was performed withthe ECL kit (Amersham Life Sciences Inc., Arlington Heights, Ill.) usingan anti-VEGFR-1 rabbit serum from Santa Cruz Biotech, Inc. (Santa Cruz,Calif.).

Results

Growth Rates of CSMC From Different-Aged Rats

Like most other cultured cells, our rat CSMC were maintained in a mediumsupplemented with 10% FBS. However, because certain experiments mayrequire the use of media containing lower concentrations of FBS, wewished to know the differences between different age groups in theirgrowth rates under different concentrations of FBS. All cells,regardless of the ages of rats from which they were derived, grew atincreasing rates with increasing FBS concentrations. At lowerconcentrations of FBS (0 and 2.5%), the growth rates showed littledifferences between different age groups. However, at higherconcentrations of FBS (5 and 10%), differences in growth rates becamemore pronounced. In particular, cells from 4-week-old rats seemed torespond best to higher concentrations of FBS and cells from 28-month-oldrats least well.

VEGF Secretion by CSMC from Different-Aged Rats

VSMC have been shown to be the principal source of secreted VEGF in thevascular (aorta) system. Pueyo, et al., Exp. Cell Res., 238: 354 (1998).We therefore examined our rat CSMC for their ability to produce VEGF. Wechose 72 hr after seeding the cells as the point of time to assay forthe secreted VEGF. Because CSMC from different age groups grew atdifferent rates, we also determined the cell numbers for each of thetested cells. When the concentrations of the secreted VEGF were adjustedfor the numbers of cells at the time of assay (FIG. 2A), it is apparentthat, within the young rat groups (ages 1 to 16 weeks), CSMC from morematured rats secreted more VEGF than CSMC from less matured rats (FIG.2C). However, CSMC from old rats (28 months old) produced similaramounts of VEGF as those from the very young rats (1 and 2 weeks old).

Effects of VEGF on Cell Growth

It has been reported that VEGF did not stimulate growth of VSMC.Grosskreutz, et al., Microvasc. Res., 58: 128 (1999). This observationwas confirmed with VSMC from 16-weeks-old rats (FIG. 3A). However, CSMCfrom all ages of rats responded to VEGF in the form of cellproliferation. Their growth rates increased with increasingconcentrations of VEGF up to 12.5 ng/ml, and after which, the cellgrowth rate started to decline with increasing concentrations of VEGF(FIG. 3B-I). When compared at the optimal dosage (12.5 ng/ml) of VEGF,all cells from young rats (1 to 16 weeks old) outgrew cells from the oldrats (28 months) and the peak growth occurred with cells from11-weeks-old rats (FIG. 3J).

Effects of VEGF on Cell Motility

It has been reported that VEGF stimulated migration of VSMC.Grosskreutz, et al., Microvasc. Res., 58: 128 (1999). This observationwas confirmed with VSMC from 16-weeks-old rats (FIG. 4A). Similarly,VEGF stimulated migration of CSMC from both young and old rats in adose-dependent manner up to the 10 ng/ml point (except the 1-week-old,which peaked at 1 ng/ml). At higher concentrations (100 and 500 ng/ml)of VEGF, the mobility of all tested cells started to decline (FIG.4B-I). When compared at the optimal dosage (10 ng/ml) of VEGF, all cellsfrom young rats (1 to 16 weeks old) out-migrated cells from the old rats(28 months) and the peak migration rate occurred with cells from4-weeks-old rats (FIG. 4J).

Identification of VEGFR-1 and VEGFR-2 mRNA Expression

The above cell proliferation and migration assay results suggested thepresence of functional VEGF receptors in CSMC. To verify this, we useRT-PCR to examine the CSMC for the expression of VEGFR-1 and VEGFR-2mRNAs. As shown in FIG. 5A, CSMC of different-aged rats expressedVEGFR-1 mRNA at different levels, being very low for the young ones (1to 3 weeks of age) and the very old (28 months of age) and high for theadolescent (4 and 6 weeks of age) and the matured (11 and 16 weeks ofage). This pattern of VEGFR-1 mRNA expression is similar to that ofVEGF-induced cell proliferation (FIG. 3).

On the other hand, CSMC, regardless of the ages of rats from which theywere derived, did not express VEGFR-2 (Lanes 1 to 8, FIG. 5B). Thisnegative result could not have been due to improper RT-PCR conditions orimproper primer design because rat heart, aorta, and penis all producedpositive results under the same experimental conditions (lanes 10, 11,and 12, FIG. 5B). The positive VEGFR-2 expression in heart, aorta, andpenis was most likely derived from vascular endothelial cells that wereincluded in the preparation of these tissue RNAs. It should be pointedout that our rat VSMC were also negative for VEGFR-2 expression (Lane 9,FIG. 5B). This is in agreement with a previous study which reported thatSMC of rat carotid arteries express VEGFR-1 but not VEGFR-2. Couper, etal., Circ. Res., 81: 932 (1997).

Identification of VEGFR-1 Protein Expression

To ascertain that VEGFR-1 protein was indeed expressed in CSMC, weperformed immunoblotting experiments using a VEGFR-1-specific antibody.As shown in FIG. 6, VEGFR-1 was detected in CSMC from rats of all ages,and the levels of its expression was very similar to those seen in theresults of RT-PCR experiments (FIG. 5A). Therefore, both the expressionof VEGFR-1 mRNA and VEGFR-1 protein correlated well with theVEGF-regulated growth rate of CSMC.

Discussion

The erectile function of the penis is that of a vascular organ. Likethose of other vascular organs, the development and growth of the penilevasculature are expected to be governed by angiogenic growth factorssuch as VEGF. Surprisingly, reports concerning VEGF expression in thepenis have been scant. In two separate studies, Burchardt et al.reported the identification of novel VEGF splice variants in the penis.Burchardt et al., Biol. Reprod., 60: 398 (1999), Burchardt, et al.,IUBMB Life, 48: 405 (1999). In another study concerning the expressionof various growth factors in the penis, Jung et al. reported theidentification of VEGF₁₈₉ mRNA in the penis. Jung, et al., Int. J.Impotence Res., 11: 247 (1999). Our ischemia rat model clearlydemonstrated the beneficial effects of VEGF on the restoration of theerectile function following surgical procedures that restricted bloodsupply to the penis.

We have conducted several experiments (RT-PCR, western blots,immunohistochemical stainings) on the expression of various VEGF formsand their receptors in the penis (unpublished). In trying to interpretthe results of those experiments, we were confronted with the questionwhich cell types (smooth muscle, endothelium, nerve, etc.) were thesource of a positive gene expression. By using cultures of a single celltype, we were able to show in the present study that CSMC expressed VEGFand VEGFR-1 but not VEGFR-2. We also showed that both the secretion ofVEGF and the expression of VEGFR-1 increased with the age of young rats(from 1 to 16 weeks of age) but declined in very old rats (28 months ofage).

We are aware of only three previously published reports that studied theeffects of VEGF on SMC or expression of VEGF receptors in SMC. First,Brown et al. showed that cultured human uterine SMC expressed bothVEGFR-1 and VEGFR-2 and responded to VEGF stimulation in the form ofcell proliferation. Brown, et al., Lab. Invest., 76: 245 (1997). Theseauthors also showed that human colon SMC did not express VEGF receptorsand did not respond to VEGF stimulation. Secondly, Couper et al.observed high levels of VEGFR-1, but no VEGFR-2, expression in SMC ofrat carotid arteries following balloon injury. Couper, et al., Circ.Res., 81: 932 (1997). Thirdly, Grosskreutz et al. showed that culturedbovine aorta SMC expressed both VEGFR-1 and VEGFR-2 and responded toVEGF stimulation in the form of cell migration (but not cellproliferation). Grosskreutz, et al., Microvasc. Res., 58: 128 (1999). Inthe present study, we used rat aorta SMC for comparison with rat CSMC.We found that, like bovine aorta SMC, rat aorta SMC responded to VEGFstimulation in the form of cell migration but not cell proliferation.However, we could only identify VEGFR-1 but not VEGFR-2 expression inrat aorta SMC. Whether the discrepancy regarding VEGFR-2 expression isdue to species difference (bovine verses rat) or other factors (age)needs to be clarified in future studies.

The significance of the proliferative and migratory responses of CSMCtoward VEGF is not known. Brown et al. speculated that alterations inexpression of VEGF or VEGF receptors may play a role in the pathogenesisof smooth muscle tumors in the uterus. Brown, et al., Lab. Invest., 76:245 (1997). Grosskreutz et al. proposed that VEGF might have achemoattractant role in the recruitment of smooth muscle cells duringthe formation of a blood vessel wall. Grosskreutz, et al., Microvasc.Res., 58: 128 (1999). Because the proliferative effect of VEGF occurredmainly with CSMC of young adult rats (11 weeks of age), it is possiblethat VEGF at least play a role in maintaining a healthy population ofCSMC. As for the migratory effect of VEGF, which occurred mainly witheven younger rats (4 weeks of age), we propose that VEGF might play arole in recruiting and/or locating CSMC to the proper sites in thecavernous spaces during adolescence.

In conclusion, we believe our present study has made the following novelobservations: (1) CSMC secreted VEGF, (2) CSMC expressed a VEGFreceptor, (3) CSMC exhibited migratory and proliferative responses toVEGF, and (4) CSMC from different-aged rats expressed different levelsof VEGF and VEGFR-1 and responded to VEGF at different rates. Liu, X.,et al., J. Urol. 166:354-360 (2001).

References

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EXAMPLE 5 The Effect of a Vascular Endothelial Growth Factor (VEGF) andAdeno-Associated Brain Derived Neurotrophic Factor (AAV-BDNF) for theTreatment of Erectile Dysfunction Induced by Hypercholesterolemia in aRat Model

Twenty-one Sprague-Dawley rats were used. All rats were fed a 2.5%cholesterol diet with added lard starting at 2 weeks of age for 6months. The rats were divided into three groups. All groups after twomonths of a high cholesterol diet underwent serum evaluation ofcholesterol and intracavernous injection of saline or treatment. Group1, the control group, received intracavernous injection of saline, group2, the VEGF group, received intracavernous injection of 4 ug of VEGF,and group 3, the AAV-BDNF group received 15 ug of BDNF. After six monthsof cholesterol diet and 4 months of treatment, all rats were subjectedto cavernous nerve electrostimulation, cavernosometry withintracavernous papaverine injection, and infusion cavernosometry withheparinized saline to measure erectile function.

Results

Serum cholesterol levels were significantly higher in animals fed thehigh cholesterol diet. Systemic arterial pressure was not significantlydifferent among the different groups. During electrostimulation of thecavernous nerve, peak sustained intracavernous pressure wassignificantly lower in the cholesterol only group (50+/−23 cm. H₂O)compared to the control and the VEFG and AAV-BDNF groups. During thepharmacologic erection phase of the cavernosometry, the VEGF and BDNFtreated groups had significantly higher sustained intracavernouspressures in comparison to the high cholesterol controls. No differencewas noted with respect to the infusion cavernosometry assessing venousleak when comparing the controls and the treated groups.

Conclusion

Rats developed erectile dysfunction after being fed a high cholesteroldiet (2.5% with lard) for 6 months. VEGF and AAV-BDNF seem to reversethe erectile dysfunction caused by high cholesterol diet.

EXAMPLE 6 The Effect of Intracavernous Vascular Endothelial GrowthFactor (VEGF) on a Rodent Model of Neurogenic Erectile Dysfunction

Objective

To test the hypothesis that intracavernous injection of VEGF canfacilitate regeneration of the cavernous nerve and restore erectilefunction after cavernous nerve injury in rats

Materials and Methods

Seventeen 3 months old Sprague Dowry rats underwent bilateral freezingof the cavernous nerves with a thermocouple immersed in liquid nitrogen.Through an abdominal incision both cavernous nerves were isolatedlateral to the prostate and frozen with thermocouple for one minutetwice (temperature cycle −130° C. to −3° C.). Minutes later,intracavernous injection of saline (n=7) or VEGF, 4 ug, (n=9) was given.Three months later, all rats underwent re-exploration andelectrostimulation of the cavernous nerves to assess erectile function.Eight additional rats underwent exploration only (sham group)

Results

The maximal intracavernous pressure in the rats underwent sham operationwas 90±8.15 cm H2O. The maximal intracavernous pressure of both thesaline treated group (39.29±5.02 cm H₂O) and the VEGF treated group(72.78±10.87 cm H₂O) were lower than the sham group. Nevertheless, theintracavernous pressure in the VEGF treated group was significantlyhigher than the saline treated group (p=0.0389).

Conclusion

Although VEGF is known as an angiogenetic factor, previous reports havesuggested that it may also be neuroprotective and neurotrophic. Ourstudy shows that intracavernous injection of VEGF significantlyfacilitated recovery of erectile function after bilateral cavernousnerve injury. If further studies confirmed that VEGF enhances bothangiogenesis and neural regeneration, intracavernous VEGF therapy may bethe treatment of choice in helping patient recover potency after radicalprostatectomy or cryoablation of the prostate.

EXAMPLE 7 Synergistic Neurotrophic Effects of Vascular EndothelialGrowth Factor and Brain-Derived Neurotrophic Factor

Neurogenic impotence due to cavernous nerve injury occurs frequently inpatients having undergone radical surgeries for prostate, bladder andrectal cancer. To investigate the feasibility of an in vitro assaysystem for nerve regeneration, we devised a model in which the majorpelvic ganglia (MPG), from which the cavernous nerves are originated,were isolated and grown in culture.

Materials and Methods

Each freshly isolated MPG was cut into 8 pieces of approximately equalsize and each piece was then attached to a Matrigel-coated coverslip.The MPG pieces were cultured in serum-free medium supplemented withphosphate-buffered saline (PBS, control), 50 ng/ml of vascularendothelial growth factor (VEGF), 20 ng/ml of brain-derived neurotrophicfactor (BDNF), or VEGF+BDNF. After 2 days of incubation, the ganglialtissues with their outgrowing nerve fibers were stained for theexpression of NADPH diaphorase and acetylcholinesterase (AchE).

Results

The average lengths of the outgrowing nerve fibers were determined to be16±7 nm, 89±21 nm, 75±11 nm and 124±23 nm, for tissues treated with PBS,VEGF, BDNF, and VEGF+BDNF, respectively (Table 10). The levels of NADPHdiaphorase expression, calculated as integrated density value (IDV),were 2,010±82, 10,126±187, 9,376±111 and 13,616±210 for tissues treatedwith PBS, VEGF, BDNF and VEGF+BDNF, respectively. The levels of AchEexpression were 29±2, 8,470±199, 1,102±211 and 11,006±198 for tissuestreated with PBS, VEGF, BDNF and VEGF+BDNF, respectively. TABLE 10Growth Average fiber NADPH staining AchE staining factor length, in nmintensity* intensity* VEGF  89 ± 21 10,126 ± 187  8,470 ± 199 BDNF  75 ±11  9,376 ± 111  1,102 ± 211 VEGF + BDNF 124 ± 23 13,616 ± 210 11,006 ±198 Control  16 ± 7  2,010 ± 82    29 ± 2 (PBS)*Expressed in Integrated Density Value (IDV).Conclusions

These results indicate that both VEGF and BDNF had neurotrophic effectsand were synergistic in combination. In addition, it appears that BDNFpreferentially promoted the growth of NOS-containing nerves (positivefor NADPH diaphorase), as opposed to cholinergic nerves (AchE-positive).

EXAMPLE 8 Synergistic Neurotrophic Effects of Vascular EndothelialGrowth Factor and Neurotrophins

We have been using several experimental systems to test the efficacy ofcombination growth factor therapies for impotence. One system employscultured rat major pelvic ganglia (MPG) that are known to innervate allurogenital organs, including the penis. We discovered that the culturedMPG are able to sprout new nerve fibers (nerve regeneration) from theaxonal ends during the first 3 days of culture, especially when treatedwith VEGF, BDNF, NT-3, or NT-4. The average lengths (in nM) of thegrowing fibers 48 hours after culturing with various treatments arelisted in Table 11. TABLE 11 GROWTH FACTORS FIBER LENGTH VEGF 89 BDNF 75NT-3 112 NT-4 81 VEGF + BDNF 124 VEGF + NT-3 132 VEGF + NT-4 101 BDNF +NT-3 88 BDNF + NT-4 81 Control (treated with saline) 16

Compared with an average length of 16 nm in control MPG, it is clearthat VEGF, BDNF, NT-3, and NT-4 all have neurotrophic effects. Moreimportantly, when VEGF is combined with each of the three neurotrophins,there was a synergistic effect.

EXAMPLE 9 VEGF-Enhanced Neurotrophin Therapy (VENT).

This example shows the testing of the hypothesis that intracavernousinjection of vascular endothelial growth factor (VEGF) enhances theeffect of brain derived neurotrophic factor (BDNF) and thus facilitateneural and erectile function recovery after cavernous nerve injury.

Materials and Methods

Three months old Sprague-Dawley rats (N=30) were used: 6 underwent asham operation; 24 underwent bilateral cavernous nerve freezing and thenrandomly divided into four groups of 6 each. The four groups of ratsreceived intracavernous injection of one of the following: 1) phosphatebuffered saline (PBS), 2) VEGF protein (4 μg), 3) adeno-associated virus(AAV)-BDNF, and 4). VEGF+AAV-BDNF. Erectile function was assessed bycavernous nerve electrostimulation 3 months later and samples of peniletissue were obtained for electron microscopy and immunohistochemicalstudies.

Results

The maximal intracavernous pressure (MICP) of the rats (mean±SEM cm H2O)in the sham, PBS, VEGF, BDNF and VEGF+BDNF groups were 100.67±6.55,29.25±3.90, 37.33±3.29, 69.75±8.54, 87.17±6.00 respectively. Thedifferences were statistically significant between the sham and allother groups but not between sham and the VEGF+BDNF group. The bloodvessel+capillary count was decreased in the PBS group (3±1.6 vs. 6±1.3in sham group) but was significant higher in the VDGF+BDNF group(9.8±2.3, p=0.01 vs. PBS group). Image analysis of nNOS stainingrevealed significantly less nNOS positive nerve fibers in the dorsalnerves in the PBS groups as compared to the sham group. There was anincreased number of positive nerve fibers in VEGF, BDNF and VEGF+BDNFgroups as compared to PBS group. Electron microscopy revealed that theVEGF+BDNF group had the best overall morphology of both myelinated andnonmyelinated nerve fibers in the penis.

Conclusions

Intracavernous injection of VEGF and AAV-BDNF improved the regenerationof nerve fibers in the penis and the recovery of erectile function. Thebest results were obtained with intracavernous injection of both VEGFand AAV-BDNF. The increased endothelial permeability of VEGF mayfacilitate the transport of itself and AAV-BDNF to the erectile tissueand thus enhance the neurotrophic and angiogenic effect.

EXAMPLE 10 A New Model for Angiogenesis Studies

Penile dorsal arteries were isolated from rats and cut into 1-mmsegments under a 10× microscope. Each segment was attached to aMatrigel-coated coverslip, which was then put in a 60-mm culture dish.Serum-free medium with or without VEGF was added and the arterialculture was incubated for 3 days. The results demonstrate the budding ofnew blood vessels that were significantly longer in VEGF-treated than inuntreated culture. The budding vessels were stained positive with RECAantibody, indicating the endothelial identity. To our best knowledge,this is the first demonstration of angiogenesis from blood vesselexplants. The ease of this experimental procedure in comparison withexisting methods should greatly facilitate future angiogenesis studies.

EXAMPLE 11 Artery-Muscle Co-Culture Systems

We extended our above angiogenesis model to include muscles in aco-culture system. The source of muscle was corpus cavernosum, urethrasphincter, or bladder. In each case, the outgrowing blood vessels showedattraction to and seemingly entering the muscle. It also appears thatVEGF encourage the growth of endothelial cells and VEGF+PDGF stimulatedthe growth of both endothelial and smooth muscle cells. In addition, thecombination of VEGF and PDGF encouraged more extensive contacts betweenthe outgrowing vessels with the muscle. More studies with either singleor a combination of other growth factors and the molecular mechanism ofvessel growth are currently underway. Once the best growth factor orcombinations are identified, we will perform in vivo studies in ratswith severe vasculogenic ED to see if the in vitro assay helps identifythe best angiogenins.

The examples set forth above are provided to give those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the preferred embodiments of the compositions, and are notintended to limit the scope of what the inventors regard as theirinvention. Modifications of the above-described modes for carrying outthe invention that are obvious to persons of skill in the art areintended to be within the scope of the following claims. Allpublications, patents, and patent applications cited in thisspecification are incorporated herein by reference as if each suchpublication, patent or patent application were specifically andindividually indicated to be incorporated herein by reference.

1. A method for preventing or treating male erectile dysfunction orfemale sexual arousal disorder induced or secondary to nerve injury,comprising administering to a mammal to whom such prevention ortreatment is needed or desirable, a combination comprising: a) aneffective amount of vascular endothelial growth factor (VEGF), whereinthe VEGF is a full length protein or a functional derivative or fragmentthereof; and b) an effective amount of a factor selected from the groupconsisting of brain-derived growth factor (BDNF), neurotrophin-3 (NT-3),and neurotrophin-4 (NT-4), wherein the factor is a full length proteinor a functional derivative or fragment thereof; thereby preventing ortreating the male erectile dysfunction or the female sexual arousaldisorder in the mammal.
 2. The method of claim 1, wherein the mammal isa human and the factor, or a functional derivative or fragment thereof,is of human origin.
 3. The method of claim 1, wherein the factorprotein, or a functional derivative or fragment thereof, is administeredby intracavernous injection, subcutaneous injection, intravenousinjection, intramuscular injection, intradermal injection, or topicaladministration.
 4. The method of claim 1, wherein the factor protein, ora functional derivative or fragment thereof, is administered via aliposome.
 5. The method of claim 1, wherein the factor protein or afunctional derivative or fragment thereof, is administered in an amountsufficient to improve blood flow and regenerate nerve and smooth musclein the clitoris and vaginal wall.
 6. The method of claim 1, wherein thefactor protein or a functional derivative or fragment thereof, isadministered in a cream or via injection to the clitoris and vaginalwall of the patient.
 7. The method of claim 1, wherein the factorprotein or a functional derivative or fragment thereof, is administeredby intracavernous injection.
 8. The method of claim 1, wherein thecombination is in the form of a pharmaceutical composition.
 9. Themethod of claim 8, wherein the combination further comprises apharmaceutically acceptable carrier or excipient.
 10. The method ofclaim 1, wherein the combination consists essentially of VEGF and BDNF.11. The method of claim 10, wherein wherein the dose of each factorprotein, or fuctional derivative or fragment thereof, in the combinationis 10-200 mcg/70 kg body weight administered once every two to sixmonths.